rnase hi domain  (Thermo Fisher)


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    Name:
    RNase H 5 U µL
    Description:
    Thermo Scientific Ribonuclease H RNase H specifically degrades the RNA strand in RNA DNA hybrids It does not hydrolyze the phosphodiester bonds within single stranded and double stranded DNA and RNA Applications• Removal of mRNA prior to synthesis of second strand cDNA• RT PCR and qRT PCR removal of RNA after first strand cDNA synthesis• Removal of the poly A sequences of mRNA after hybridization with oligo dT • Site specific cleavage of RNA• Studies of in vitro polyadenylation reaction products
    Catalog Number:
    en0201
    Price:
    None
    Applications:
    One-Step qRT-PCR|PCR & Real-Time PCR|RT-PCR|Real Time PCR (qPCR)|Reverse Transcription|Two-Step RT-PCR
    Category:
    Proteins Enzymes Peptides
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    Structured Review

    Thermo Fisher rnase hi domain
    Crystals of the <t>RNase</t> HI <t>domain-MBP</t> fusion protein formed in 20% PEG 2000 and 0.2 M diammonium tartrate.
    Thermo Scientific Ribonuclease H RNase H specifically degrades the RNA strand in RNA DNA hybrids It does not hydrolyze the phosphodiester bonds within single stranded and double stranded DNA and RNA Applications• Removal of mRNA prior to synthesis of second strand cDNA• RT PCR and qRT PCR removal of RNA after first strand cDNA synthesis• Removal of the poly A sequences of mRNA after hybridization with oligo dT • Site specific cleavage of RNA• Studies of in vitro polyadenylation reaction products
    https://www.bioz.com/result/rnase hi domain/product/Thermo Fisher
    Average 85 stars, based on 32804 article reviews
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    rnase hi domain - by Bioz Stars, 2020-08
    85/100 stars

    Images

    1) Product Images from "Cloning, expression, purification and preliminary crystallographic analysis of the RNase HI domain of the Mycobacterium tuberculosis protein Rv2228c as a maltose-binding protein fusion"

    Article Title: Cloning, expression, purification and preliminary crystallographic analysis of the RNase HI domain of the Mycobacterium tuberculosis protein Rv2228c as a maltose-binding protein fusion

    Journal: Acta Crystallographica Section F: Structural Biology and Crystallization Communications

    doi: 10.1107/S1744309108021118

    Crystals of the RNase HI domain-MBP fusion protein formed in 20% PEG 2000 and 0.2 M diammonium tartrate.
    Figure Legend Snippet: Crystals of the RNase HI domain-MBP fusion protein formed in 20% PEG 2000 and 0.2 M diammonium tartrate.

    Techniques Used:

    SDS–PAGE gel showing total cell extract (lane A ), soluble fraction (lane B ) and insoluble fraction (lane C ) from IPTG-based induction of the RNase HI-MBP fusion protein in pMAL-C2.
    Figure Legend Snippet: SDS–PAGE gel showing total cell extract (lane A ), soluble fraction (lane B ) and insoluble fraction (lane C ) from IPTG-based induction of the RNase HI-MBP fusion protein in pMAL-C2.

    Techniques Used: SDS Page

    2) Product Images from "RNase HII saves rnhA mutant Escherichia coli from R-loop-associated chromosomal fragmentation"

    Article Title: RNase HII saves rnhA mutant Escherichia coli from R-loop-associated chromosomal fragmentation

    Journal: Journal of molecular biology

    doi: 10.1016/j.jmb.2017.08.004

    Chromosome fragmentation analysis by RNase HI, RNase HII and RNase A treatment in vitro A. A scheme of various hypothetical R-lesions (R-tract, two types of R-gaps) with positions of cleavage by RNase HI, HII and A (in low salt (LS) and high salt (HS) conditions) shown with arrows of the corresponding color. Small blue “d” letters, dNs; small orange “r” letters, rNs. The strand polarity in a duplex is identified on the left. B. A representative pulsed-field gel detecting chromosomal fragmentation after RNase HII treatment. The lanes are marked either with “b” (buffer treatment control) or “H2” (RNase HII treatment). Strains: WT, AB1157; rnhA , L-413; rnhB , L-415; rnhAB , L-416; uvrA rnhAB , L-417. C. Quantification of the RNase treatment-induced fragmentation. The plotted values are means ± SEM from 3-6 independent measurements from gels like in “B”. For RNase A treatment, both low salt (LS) and high salt (HS) conditions are plotted. Since individual fragmentation values are differences between the enzyme and the buffer treatments, some values are negative.
    Figure Legend Snippet: Chromosome fragmentation analysis by RNase HI, RNase HII and RNase A treatment in vitro A. A scheme of various hypothetical R-lesions (R-tract, two types of R-gaps) with positions of cleavage by RNase HI, HII and A (in low salt (LS) and high salt (HS) conditions) shown with arrows of the corresponding color. Small blue “d” letters, dNs; small orange “r” letters, rNs. The strand polarity in a duplex is identified on the left. B. A representative pulsed-field gel detecting chromosomal fragmentation after RNase HII treatment. The lanes are marked either with “b” (buffer treatment control) or “H2” (RNase HII treatment). Strains: WT, AB1157; rnhA , L-413; rnhB , L-415; rnhAB , L-416; uvrA rnhAB , L-417. C. Quantification of the RNase treatment-induced fragmentation. The plotted values are means ± SEM from 3-6 independent measurements from gels like in “B”. For RNase A treatment, both low salt (LS) and high salt (HS) conditions are plotted. Since individual fragmentation values are differences between the enzyme and the buffer treatments, some values are negative.

    Techniques Used: In Vitro, Pulsed-Field Gel

    Growth, morphology and viability of the double rnhAB mutants A. A scheme of in vivo substrates of the two RNase H enzymes. The common substrate, framed in bright green, is the RNA-run with at least four contiguous rNs, which we call “R-tract”. HI and H1, HII and H2 refer to RNase H enzymes of prokaryotes and eukaryotes accordingly. B. Colony size on LB agar, 37°C, 24 hours. Strains: WT, AB1157; Δ rnhA , L-413; Δ rnhB , L-415; Δ rnhAB , L-416. C. Images of rnh and wild type strains stained with DAPI and observed by Hiraga's fluo-phase combined method. Cells were grown at 37°C in LB. The strains are like in “B”. D. Viability of the strains, determined as the ratio of the colony forming units (CFUs) to the microscopic counts in the same volume of the culture. Overnight cultures grown at 30°C were diluted and grown at the temperature (indicated by the first number) to OD 0.2-0.3 (about 2 hours), then cultures were serially diluted and plated on LB plates developed for 16 hours at the temperature indicated by the second number in pairs. Average viability (± SEM) of the eight WT measurements and six measurements for the rnhAB mutant cells is shown (the low titers of the two MG1655 Δ rnhAB cultures at 42°C were not used in the calculation). Strains: AB1157, L-416, MG1655, L-419. E. An enlarged image of the rnhAB mutant cells (processed as in panel C), to show nucleoids of both filamenting and normal-looking cells in some detail. F. Anaerobic growth inhibition of the rnhA and anaerobic lethality of rnhAB strains. Dilution-spotting of strains (like in “B”) was done in an anaerobic chamber on LB plates. Plates were incubated at room temperature in the chamber for 24 hours, then shifted to 28°C aerobic conditions for another 48 hours. G. The uvrA defect further reduces the colony size of the rnhAB double mutant. Strains: rnhAB , L-416; uvrA rnhA , L-414; uvrA rnhAB , L-417.
    Figure Legend Snippet: Growth, morphology and viability of the double rnhAB mutants A. A scheme of in vivo substrates of the two RNase H enzymes. The common substrate, framed in bright green, is the RNA-run with at least four contiguous rNs, which we call “R-tract”. HI and H1, HII and H2 refer to RNase H enzymes of prokaryotes and eukaryotes accordingly. B. Colony size on LB agar, 37°C, 24 hours. Strains: WT, AB1157; Δ rnhA , L-413; Δ rnhB , L-415; Δ rnhAB , L-416. C. Images of rnh and wild type strains stained with DAPI and observed by Hiraga's fluo-phase combined method. Cells were grown at 37°C in LB. The strains are like in “B”. D. Viability of the strains, determined as the ratio of the colony forming units (CFUs) to the microscopic counts in the same volume of the culture. Overnight cultures grown at 30°C were diluted and grown at the temperature (indicated by the first number) to OD 0.2-0.3 (about 2 hours), then cultures were serially diluted and plated on LB plates developed for 16 hours at the temperature indicated by the second number in pairs. Average viability (± SEM) of the eight WT measurements and six measurements for the rnhAB mutant cells is shown (the low titers of the two MG1655 Δ rnhAB cultures at 42°C were not used in the calculation). Strains: AB1157, L-416, MG1655, L-419. E. An enlarged image of the rnhAB mutant cells (processed as in panel C), to show nucleoids of both filamenting and normal-looking cells in some detail. F. Anaerobic growth inhibition of the rnhA and anaerobic lethality of rnhAB strains. Dilution-spotting of strains (like in “B”) was done in an anaerobic chamber on LB plates. Plates were incubated at room temperature in the chamber for 24 hours, then shifted to 28°C aerobic conditions for another 48 hours. G. The uvrA defect further reduces the colony size of the rnhAB double mutant. Strains: rnhAB , L-416; uvrA rnhA , L-414; uvrA rnhAB , L-417.

    Techniques Used: In Vivo, Staining, Mutagenesis, Inhibition, Incubation

    Verification of RNase HI and RNase HII rN-DNA substrate specificity in vitro and the rN-density in DNA of the RNase H + cells and rnh mutants A. A scheme of the two double stranded oligo substrates: 38R1 (single rN) and 34R5 (five consecutive rN). The 32 P label at the 5′ end is shown as a red asterisk. DNA nucleotides are shown as blue lower case “d”, ribonucleotides are orange uppercase “R”. B. Products of the rN-DNA substrate hydrolysis by E. coli RNase HI and RNase HII enzymes. The radiolabelled rN-containing dsDNA oligos (shown in A) were incubated with the RNase HI or RNase HII enzymes. “0.1 M NaOH” and “Na Carb. pH 9.3” refer to alkali conditions in which rN hydrolysis produces reference size products. Numbers “1” or “5” refer to 38R1 or 34R5 oligos (A); ss/ds refers to whether the substrate used in the reaction was single-stranded or double-stranded. RNase H1 and RNase H2 were the E. coli enzymes RNase HI and RNase HII. RNase H1-1 and RNase H1-2 were RNase HI enzymes from different producers. The numbers on the side of the gel represent the sizes of the substrate and cleavage products. The reaction products were analyzed in 18% urea-PAGE gel. C. Only 34R5 oligo was used as either ss or ds substrate. All designations are like in “B”. D. Treatment with RNase HII of the plasmid isolated by alkaline lysis protocol. SCM, supercoiled monomer; b, buffer; H2, RNase HII. Plasmid: pEAK86, plasmid isolation was done at 0°C. Strains for results shown in panels D-I were: WT, AB1157; rnhA , L-413; rnhB , L-415; rnhAB , L-416; uvrA rnhAB L-417. Product of the reactions were run in 1.1% agarose gel; autoradiogram of the representative Southern blot with the radiolabelled pEAK86 DNA as a probe is shown here and also in E and G. E. Treatment with either RNase HI or RNase HII enzymes of the plasmid isolated by the total genomic DNA protocol. SC, supercoiled plasmid; relaxed, relaxed plasmid; chrom., chromosomal DNA. Plasmid: pEAK86. Analysis of plasmid species was carried out as in D. F. Summary of quantification of the RNaseHII-revealed density of rNs in plasmid DNA isolated by various methods from the rnhAB double mutant. The density calculations are described in Methods. “Form.”, formamide. G. Alkali treatment analysis of rN-density. The plasmid DNA isolated by alkaline lysis at 0°C, was linearized and treated with NaOH. Treatment: “—”, no treatment; 0°, 0.2 M NaOH, 20 mM EDTA treatment on ice for 20 min; 45°, 0.3 M NaOH, 20 mM EDTA treatment at 45°C for 90 minutes. ds, linearized plasmid DNA, ss -single stranded plasmid. The samples were run in 1.1% agarose in TAE buffer, at 4°C. H. Summary of quantification of the rN-density determined by either RNase HII or by alkali treatments (from gels like in “G”). Various mutant comparison data are shown, pEAK86 was purified by alkaline lysis only, values are means of three independent measurements ± SEM. The star identifies the value already reported in panel “F”. I. R-loop removal by RNase HI or by RNase A. pAM34 isolated from rnhA (strain L-413) by the total genomic DNA protocol.
    Figure Legend Snippet: Verification of RNase HI and RNase HII rN-DNA substrate specificity in vitro and the rN-density in DNA of the RNase H + cells and rnh mutants A. A scheme of the two double stranded oligo substrates: 38R1 (single rN) and 34R5 (five consecutive rN). The 32 P label at the 5′ end is shown as a red asterisk. DNA nucleotides are shown as blue lower case “d”, ribonucleotides are orange uppercase “R”. B. Products of the rN-DNA substrate hydrolysis by E. coli RNase HI and RNase HII enzymes. The radiolabelled rN-containing dsDNA oligos (shown in A) were incubated with the RNase HI or RNase HII enzymes. “0.1 M NaOH” and “Na Carb. pH 9.3” refer to alkali conditions in which rN hydrolysis produces reference size products. Numbers “1” or “5” refer to 38R1 or 34R5 oligos (A); ss/ds refers to whether the substrate used in the reaction was single-stranded or double-stranded. RNase H1 and RNase H2 were the E. coli enzymes RNase HI and RNase HII. RNase H1-1 and RNase H1-2 were RNase HI enzymes from different producers. The numbers on the side of the gel represent the sizes of the substrate and cleavage products. The reaction products were analyzed in 18% urea-PAGE gel. C. Only 34R5 oligo was used as either ss or ds substrate. All designations are like in “B”. D. Treatment with RNase HII of the plasmid isolated by alkaline lysis protocol. SCM, supercoiled monomer; b, buffer; H2, RNase HII. Plasmid: pEAK86, plasmid isolation was done at 0°C. Strains for results shown in panels D-I were: WT, AB1157; rnhA , L-413; rnhB , L-415; rnhAB , L-416; uvrA rnhAB L-417. Product of the reactions were run in 1.1% agarose gel; autoradiogram of the representative Southern blot with the radiolabelled pEAK86 DNA as a probe is shown here and also in E and G. E. Treatment with either RNase HI or RNase HII enzymes of the plasmid isolated by the total genomic DNA protocol. SC, supercoiled plasmid; relaxed, relaxed plasmid; chrom., chromosomal DNA. Plasmid: pEAK86. Analysis of plasmid species was carried out as in D. F. Summary of quantification of the RNaseHII-revealed density of rNs in plasmid DNA isolated by various methods from the rnhAB double mutant. The density calculations are described in Methods. “Form.”, formamide. G. Alkali treatment analysis of rN-density. The plasmid DNA isolated by alkaline lysis at 0°C, was linearized and treated with NaOH. Treatment: “—”, no treatment; 0°, 0.2 M NaOH, 20 mM EDTA treatment on ice for 20 min; 45°, 0.3 M NaOH, 20 mM EDTA treatment at 45°C for 90 minutes. ds, linearized plasmid DNA, ss -single stranded plasmid. The samples were run in 1.1% agarose in TAE buffer, at 4°C. H. Summary of quantification of the rN-density determined by either RNase HII or by alkali treatments (from gels like in “G”). Various mutant comparison data are shown, pEAK86 was purified by alkaline lysis only, values are means of three independent measurements ± SEM. The star identifies the value already reported in panel “F”. I. R-loop removal by RNase HI or by RNase A. pAM34 isolated from rnhA (strain L-413) by the total genomic DNA protocol.

    Techniques Used: In Vitro, Incubation, Polyacrylamide Gel Electrophoresis, Plasmid Preparation, Isolation, Alkaline Lysis, Agarose Gel Electrophoresis, Southern Blot, Mutagenesis, Purification

    3) Product Images from "Hsp90 protein interacts with phosphorothioate oligonucleotides containing hydrophobic 2′-modifications and enhances antisense activity"

    Article Title: Hsp90 protein interacts with phosphorothioate oligonucleotides containing hydrophobic 2′-modifications and enhances antisense activity

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw144

    Hsp90 protein binds PS/cEt or PS/LNA ASOs but not PS/MOE ASO. ( A ) Affinity selection was performed using biotinylated gapmer PS-ASOs with different 2′-modifications in the wings. Proteins were eluted using non-biotinylated PS-ASOs with the same chemical composition. Isolated proteins were separated by SDS-PAGE, and visualized by silver staining (upper panel), or detected by Western assay for a duplicate gel (lower panel) using antibodies specific to Hsp90α or HSP90β. Ku70 protein served as a control for loading. The protein band containing Hsp90 is indicated for the silver stained gel. ( B ) Western analysis for Hsp90 protein co-selected using a biotinylated PS/cEt ASO, and eluted by competition using PS/cEt ASOs with two different sequences that target PTEN or NCL1 mRNAs, or using a PS/MOE ASO targeting NCL1. The Hsp90 antibody recognizes both α and β isoforms. Ku70 protein was detected and served as a control. ( C ) Affinity selection using PS/cEt ASOs that have the same sequence and chemistry but tagged with biotin at either 5′ or 3′ end. Co-isolated Hsp90 and Ku70 were detected by western assay. ( D ) Affinity selection was carried out using either a single stranded PS/cEt ASO or a duplex formed using the same ASO and a complementary 2′- O -methylated oligonucleotide. Co-isolated proteins were directly separated on SDS-PAGE and analyzed by Western assay for Hsp90 and Ku70 proteins. ( E ) Affinity selection was conducted using either a PS/MOE ASO 386652 or a PS/cEt ASO 586183. After washing, bound proteins were first eluted by RNase I (5 U/μl) or DNase I (5 U/μl) treatment for 30 min at RT, followed by elution using 9M Urea from the same beads for 30 min at RT. The eluted proteins were precipitated and analyzed by Western assay for Hsp90 protein. ( F ) Silver staining of one μg of the recombinant Hsp90α protein (Abcam, ab80369). ( G ) Interaction of the recombinant Hsp90α protein with PS/cEt ASO was determined by affinity selection using biotinylated PS-ASOs and 3 μg of purified Hsp90 protein. After wash, the beads were directly loaded on a 4–12% SDS-PAGE, and Hsp90 protein was detected by Western assay. The ASO ID numbers in each panel are shown. The above experiments were repeated at least three times and representative results are shown. ( H ) Membrane binding assay for Hsp90α protein and different PS-ASOs, as described in Materials and Methods. For each ASO, triplicate binding reactions were performed. The molar ratio between Hsp90a protein and ASO was given below the wells. An example of a double-filter binding for PS/cEt and PS/LNA ASOs is shown in the middle panel for protein-bound ASOs captured using a Hybond ECL membrane, and the lower panel for unbound ASOs captured using a Hybond-N+ nylon membrane. The signal intensity for the ASOs was quantified and the binding curves for ( I ) PS/cEt and ( J ) PS/LNA ASOs were plotted using Prism. The calculated kds are given. The error bars represent standard deviations from three experiments.
    Figure Legend Snippet: Hsp90 protein binds PS/cEt or PS/LNA ASOs but not PS/MOE ASO. ( A ) Affinity selection was performed using biotinylated gapmer PS-ASOs with different 2′-modifications in the wings. Proteins were eluted using non-biotinylated PS-ASOs with the same chemical composition. Isolated proteins were separated by SDS-PAGE, and visualized by silver staining (upper panel), or detected by Western assay for a duplicate gel (lower panel) using antibodies specific to Hsp90α or HSP90β. Ku70 protein served as a control for loading. The protein band containing Hsp90 is indicated for the silver stained gel. ( B ) Western analysis for Hsp90 protein co-selected using a biotinylated PS/cEt ASO, and eluted by competition using PS/cEt ASOs with two different sequences that target PTEN or NCL1 mRNAs, or using a PS/MOE ASO targeting NCL1. The Hsp90 antibody recognizes both α and β isoforms. Ku70 protein was detected and served as a control. ( C ) Affinity selection using PS/cEt ASOs that have the same sequence and chemistry but tagged with biotin at either 5′ or 3′ end. Co-isolated Hsp90 and Ku70 were detected by western assay. ( D ) Affinity selection was carried out using either a single stranded PS/cEt ASO or a duplex formed using the same ASO and a complementary 2′- O -methylated oligonucleotide. Co-isolated proteins were directly separated on SDS-PAGE and analyzed by Western assay for Hsp90 and Ku70 proteins. ( E ) Affinity selection was conducted using either a PS/MOE ASO 386652 or a PS/cEt ASO 586183. After washing, bound proteins were first eluted by RNase I (5 U/μl) or DNase I (5 U/μl) treatment for 30 min at RT, followed by elution using 9M Urea from the same beads for 30 min at RT. The eluted proteins were precipitated and analyzed by Western assay for Hsp90 protein. ( F ) Silver staining of one μg of the recombinant Hsp90α protein (Abcam, ab80369). ( G ) Interaction of the recombinant Hsp90α protein with PS/cEt ASO was determined by affinity selection using biotinylated PS-ASOs and 3 μg of purified Hsp90 protein. After wash, the beads were directly loaded on a 4–12% SDS-PAGE, and Hsp90 protein was detected by Western assay. The ASO ID numbers in each panel are shown. The above experiments were repeated at least three times and representative results are shown. ( H ) Membrane binding assay for Hsp90α protein and different PS-ASOs, as described in Materials and Methods. For each ASO, triplicate binding reactions were performed. The molar ratio between Hsp90a protein and ASO was given below the wells. An example of a double-filter binding for PS/cEt and PS/LNA ASOs is shown in the middle panel for protein-bound ASOs captured using a Hybond ECL membrane, and the lower panel for unbound ASOs captured using a Hybond-N+ nylon membrane. The signal intensity for the ASOs was quantified and the binding curves for ( I ) PS/cEt and ( J ) PS/LNA ASOs were plotted using Prism. The calculated kds are given. The error bars represent standard deviations from three experiments.

    Techniques Used: Allele-specific Oligonucleotide, Selection, Isolation, SDS Page, Silver Staining, Western Blot, Staining, Sequencing, Methylation, Recombinant, Purification, Binding Assay

    4) Product Images from "TDP-43 deficiency links Amyotrophic Lateral Sclerosis with R-loop homeostasis and R loop-mediated DNA damage"

    Article Title: TDP-43 deficiency links Amyotrophic Lateral Sclerosis with R-loop homeostasis and R loop-mediated DNA damage

    Journal: bioRxiv

    doi: 10.1101/2020.05.10.086652

    TDP-43 associates with FANCD2 and affects genome integrity in stable transfected SHSY-TDP382-. A) Detection of γH2AX foci, by IF in SH-SY5Y, SH-TDP+ and SH-TDP382. The histogram shows the quantification of the relative amount of cells in percentage containing > 10 γH2AX foci in each case. Details as in Fig 2A . *, P
    Figure Legend Snippet: TDP-43 associates with FANCD2 and affects genome integrity in stable transfected SHSY-TDP382-. A) Detection of γH2AX foci, by IF in SH-SY5Y, SH-TDP+ and SH-TDP382. The histogram shows the quantification of the relative amount of cells in percentage containing > 10 γH2AX foci in each case. Details as in Fig 2A . *, P

    Techniques Used: Transfection

    R-loops accumulation in SHSY-TDP382- and increased dsRNAs levels in SHSY-TDP+. A) IF using anti S9.6 antibody and anti-nucleolin antibody of SH-SY5Y, SH-TDP+ and SH-TDP382. The graph shows the quantification of S9.6 nucleolar signal. Details as in Fig 1A . ****, P
    Figure Legend Snippet: R-loops accumulation in SHSY-TDP382- and increased dsRNAs levels in SHSY-TDP+. A) IF using anti S9.6 antibody and anti-nucleolin antibody of SH-SY5Y, SH-TDP+ and SH-TDP382. The graph shows the quantification of S9.6 nucleolar signal. Details as in Fig 1A . ****, P

    Techniques Used:

    5) Product Images from "Ribonuclease H1-dependent hepatotoxicity caused by locked nucleic acid-modified gapmer antisense oligonucleotides"

    Article Title: Ribonuclease H1-dependent hepatotoxicity caused by locked nucleic acid-modified gapmer antisense oligonucleotides

    Journal: Scientific Reports

    doi: 10.1038/srep30377

    Effect of RNaseH1 knockdown on Acsl1 gapmer-derived hepatotoxicity. Acsl1 gapmer (10 mg/kg) was subcutaneously administrated 2 days after injection of siRNA-invivofectamine complex, and the liver and plasma were isolated on day 10 after ASO administration. Plasma ALT level ( a ); plasma AST level ( b ); expression level of RnaseH1 mRNA ( c ); Western blot analysis of RnaseH1 and Acsl1 protein ( d ). Two homogenates of liver in each group were analysed using β-actin as internal standard. Expression level of RnaseH1 mRNA ( e ) and RnaseH2a mRNA ( f ). In situ detection of apoptosis ( g ). Liver sections of mice that received both siRNA and Acsl1 gapmer was subjected to TUNEL assay and observed under confocal laser microscopy (TM; transmission, Scale bar: 5 μm). (n = 4, mean + S.D., * p
    Figure Legend Snippet: Effect of RNaseH1 knockdown on Acsl1 gapmer-derived hepatotoxicity. Acsl1 gapmer (10 mg/kg) was subcutaneously administrated 2 days after injection of siRNA-invivofectamine complex, and the liver and plasma were isolated on day 10 after ASO administration. Plasma ALT level ( a ); plasma AST level ( b ); expression level of RnaseH1 mRNA ( c ); Western blot analysis of RnaseH1 and Acsl1 protein ( d ). Two homogenates of liver in each group were analysed using β-actin as internal standard. Expression level of RnaseH1 mRNA ( e ) and RnaseH2a mRNA ( f ). In situ detection of apoptosis ( g ). Liver sections of mice that received both siRNA and Acsl1 gapmer was subjected to TUNEL assay and observed under confocal laser microscopy (TM; transmission, Scale bar: 5 μm). (n = 4, mean + S.D., * p

    Techniques Used: Derivative Assay, Injection, Isolation, Allele-specific Oligonucleotide, AST Assay, Expressing, Western Blot, In Situ, Mouse Assay, TUNEL Assay, Microscopy, Transmission Assay

    Effect of RNaseH1 knockdown for GR gapmer-derived hepatotoxicity. Mice were administrated an siRNA–invivofectamine complex 2 days prior to s . c . injection of 10 mg/kg gapmer. Plasma was collected 4, 7 and 10 days later, and mouse liver was isolated on day 10. ( a ) Plasma ALT level; ( b ) plasma AST level; ( c ) Expression level of RnaseH1 and ( d ) GR mRNA in liver (n = 4, mean ± S.D., * p
    Figure Legend Snippet: Effect of RNaseH1 knockdown for GR gapmer-derived hepatotoxicity. Mice were administrated an siRNA–invivofectamine complex 2 days prior to s . c . injection of 10 mg/kg gapmer. Plasma was collected 4, 7 and 10 days later, and mouse liver was isolated on day 10. ( a ) Plasma ALT level; ( b ) plasma AST level; ( c ) Expression level of RnaseH1 and ( d ) GR mRNA in liver (n = 4, mean ± S.D., * p

    Techniques Used: Derivative Assay, Mouse Assay, Injection, Isolation, AST Assay, Expressing

    Effect of RNaseH1 knockdown on other hepatotoxic gapmers. Mice that had received siRNA–invivofectamine complex were administrated hepatotoxic gapmers targeting ApoB (20 mg/kg), Hprt1 (80 mg/kg), or human Kif11 (80 mg/kg). Upper panels show plasma ALT; lower panels show plasma AST. ApoB ( a , d ), Hprt1 ( b , e ), and human Kif11 ( c , f ). (n = 3, mean ± S.D.) # Mouse was euthanized because of acute weight loss.
    Figure Legend Snippet: Effect of RNaseH1 knockdown on other hepatotoxic gapmers. Mice that had received siRNA–invivofectamine complex were administrated hepatotoxic gapmers targeting ApoB (20 mg/kg), Hprt1 (80 mg/kg), or human Kif11 (80 mg/kg). Upper panels show plasma ALT; lower panels show plasma AST. ApoB ( a , d ), Hprt1 ( b , e ), and human Kif11 ( c , f ). (n = 3, mean ± S.D.) # Mouse was euthanized because of acute weight loss.

    Techniques Used: Mouse Assay, AST Assay

    6) Product Images from "Dynamic nucleoplasmic and nucleolar localization of mammalian RNase H1 in response to RNAP I transcriptional R-loops"

    Article Title: Dynamic nucleoplasmic and nucleolar localization of mammalian RNase H1 in response to RNAP I transcriptional R-loops

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx710

    RNase H1 localizes to nucleoli in a RNAP I transcription-dependent manner. (A and B) Representative confocal immunofluorescence images of HeLa cells infected with AdV [H1] for 24 h. Cells were stained with ( A ) an antibody raised in mice against amino acids 189–287 of human RNase H1 and an anti-NPM1 antibody or ( B ) with an antibody raised in rabbit against amino acids 47–287 of human RNase H1 and an anti-BrdU antibody. Cells in panel B were pulsed with 1 mM BrU for 30 min immediately prior to fixation. ( C ) Representative confocal immunofluorescence images of HeLa cells transiently transfected with the pRNase H1-Flag for 48 h and stained using an anti-Flag antibody. ( D ) Representative confocal microscopy images of HeLa cells stained for endogenous RNase H1 with signal amplified using TSA. ( E ) Levels of 47S pre-rRNA and NCL1 pre-mRNA were quantified using qRT-PCR in extracts of HeLa cells treated with 0.02 μg/ml ActD for 2 h, 250 nM CX5461 for 6 h, or vehicle (DMSO). The error bars represent standard deviation from three parallel experiments. ( F ) HeLa cells infected with AdV [H1] for 24 h were treated with DMSO, 0.02 μg/ml ActD for 2 h, or 250 nM CX5461 for 6 h. Cells were stained with the anti-RNase H1 antibody.
    Figure Legend Snippet: RNase H1 localizes to nucleoli in a RNAP I transcription-dependent manner. (A and B) Representative confocal immunofluorescence images of HeLa cells infected with AdV [H1] for 24 h. Cells were stained with ( A ) an antibody raised in mice against amino acids 189–287 of human RNase H1 and an anti-NPM1 antibody or ( B ) with an antibody raised in rabbit against amino acids 47–287 of human RNase H1 and an anti-BrdU antibody. Cells in panel B were pulsed with 1 mM BrU for 30 min immediately prior to fixation. ( C ) Representative confocal immunofluorescence images of HeLa cells transiently transfected with the pRNase H1-Flag for 48 h and stained using an anti-Flag antibody. ( D ) Representative confocal microscopy images of HeLa cells stained for endogenous RNase H1 with signal amplified using TSA. ( E ) Levels of 47S pre-rRNA and NCL1 pre-mRNA were quantified using qRT-PCR in extracts of HeLa cells treated with 0.02 μg/ml ActD for 2 h, 250 nM CX5461 for 6 h, or vehicle (DMSO). The error bars represent standard deviation from three parallel experiments. ( F ) HeLa cells infected with AdV [H1] for 24 h were treated with DMSO, 0.02 μg/ml ActD for 2 h, or 250 nM CX5461 for 6 h. Cells were stained with the anti-RNase H1 antibody.

    Techniques Used: Immunofluorescence, Infection, Staining, Mouse Assay, Transfection, Confocal Microscopy, Amplification, Quantitative RT-PCR, Standard Deviation

    Top1 and RPA194 co-migrate to perinucleolar LNCs upon RNAP I transcriptional arrest and nucleolar segregation. ( A ) Confocal immunofluorescence imaging of HeLa cells 48 h after transfection with luciferase siRNA or Top1 siRNA. Cells were stained for Top1 and nucleolar marker NPM1. ( B ) HeLa cells treated with 250 nM CX5461 for 6 h were stained for NPM1. ( C ) Co-immunofluorescent staining of Top1 and RPA194 in HeLa cells treated with DMSO or 250 nM CX5461 (6 h).
    Figure Legend Snippet: Top1 and RPA194 co-migrate to perinucleolar LNCs upon RNAP I transcriptional arrest and nucleolar segregation. ( A ) Confocal immunofluorescence imaging of HeLa cells 48 h after transfection with luciferase siRNA or Top1 siRNA. Cells were stained for Top1 and nucleolar marker NPM1. ( B ) HeLa cells treated with 250 nM CX5461 for 6 h were stained for NPM1. ( C ) Co-immunofluorescent staining of Top1 and RPA194 in HeLa cells treated with DMSO or 250 nM CX5461 (6 h).

    Techniques Used: Immunofluorescence, Imaging, Transfection, Luciferase, Staining, Marker

    7) Product Images from "RNase H1 Regulates Mitochondrial Transcription and Translation via the Degradation of 7S RNA"

    Article Title: RNase H1 Regulates Mitochondrial Transcription and Translation via the Degradation of 7S RNA

    Journal: Frontiers in Genetics

    doi: 10.3389/fgene.2019.01393

    Mitochondrial DNA maintenance in mutant RNASEH1 fibroblasts. (A) Southern blot of total DNA digested with Pvu II from control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium. A radioactive probe against mtDNA was used to detect both linearized mtDNA (empty arrowhead) and 7S DNA (filled arrowhead and bracket), while a probe against 18S rDNA was used as loading control. (B) Relative mitochondrial DNA copy number in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium, calculated as the linearized mtDNA/18S rDNA signal ratio. Data are shown as mean ± SD, n = 4, ***p
    Figure Legend Snippet: Mitochondrial DNA maintenance in mutant RNASEH1 fibroblasts. (A) Southern blot of total DNA digested with Pvu II from control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium. A radioactive probe against mtDNA was used to detect both linearized mtDNA (empty arrowhead) and 7S DNA (filled arrowhead and bracket), while a probe against 18S rDNA was used as loading control. (B) Relative mitochondrial DNA copy number in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium, calculated as the linearized mtDNA/18S rDNA signal ratio. Data are shown as mean ± SD, n = 4, ***p

    Techniques Used: Mutagenesis, Southern Blot

    RNASEH1 mutations, transcript, and protein levels. (A) Domains ofhuman RNAse H1 protein (MTS, mitochondrial targeting sequence; HBD, hybrid binding domain; CD, connection domain; catalytic domain). RNase H1 protein sequences from representative species, H. sapiens (Hs, NP_002927) M. musculus (Mm, NP_035405), B. taurus (Bt, NP_001039970), G. gallus (Gg, NP_990329), X. tropicalis (Xt, NP_001096299), D. rerio (Dr, NP_001002659), C. intestinalis (Ci, F6QPH0), D. melanogaster (Dm, NP_995777), C. elegans (Ce, NP_001040786), S. cerevisiae (Sc, Q04740), were extracted from the database and aligned using ClustalW2. Conserved residues found mutated in the patient in exon 4 are boxed in red, while residues in the active site and interacting with DNA or RNA are boxed in yellow and green, respectively. Positions of β strands are marked by blue arrows. (B) Human RNase H1 crystal structure (PDB ID 2QK9) 18 bp DNA(cyan): RNA (magenta) hybrid is shown respectively. Residues in the active site are colored in yellow. Residues Trp 164 (green) and Val 142 , or the mutated variant Ile 142 (red), are shown as sticks. (C) RNASEH1 transcript levels in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium assessed by qPCR (probe Hs00268000_m1) and normalized to GAPDH transcript levels. Data are shown as mean ± SD, n = 4, ***p
    Figure Legend Snippet: RNASEH1 mutations, transcript, and protein levels. (A) Domains ofhuman RNAse H1 protein (MTS, mitochondrial targeting sequence; HBD, hybrid binding domain; CD, connection domain; catalytic domain). RNase H1 protein sequences from representative species, H. sapiens (Hs, NP_002927) M. musculus (Mm, NP_035405), B. taurus (Bt, NP_001039970), G. gallus (Gg, NP_990329), X. tropicalis (Xt, NP_001096299), D. rerio (Dr, NP_001002659), C. intestinalis (Ci, F6QPH0), D. melanogaster (Dm, NP_995777), C. elegans (Ce, NP_001040786), S. cerevisiae (Sc, Q04740), were extracted from the database and aligned using ClustalW2. Conserved residues found mutated in the patient in exon 4 are boxed in red, while residues in the active site and interacting with DNA or RNA are boxed in yellow and green, respectively. Positions of β strands are marked by blue arrows. (B) Human RNase H1 crystal structure (PDB ID 2QK9) 18 bp DNA(cyan): RNA (magenta) hybrid is shown respectively. Residues in the active site are colored in yellow. Residues Trp 164 (green) and Val 142 , or the mutated variant Ile 142 (red), are shown as sticks. (C) RNASEH1 transcript levels in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium assessed by qPCR (probe Hs00268000_m1) and normalized to GAPDH transcript levels. Data are shown as mean ± SD, n = 4, ***p

    Techniques Used: Sequencing, Binding Assay, Variant Assay, Real-time Polymerase Chain Reaction

    Mitochondrial transcript levels in mutant RNASEH1 fibroblasts. Mitochondrial transcript levels in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium, assessed by qPCR and normalized to GAPDH transcript levels. Analyzed transcripts included the non-coding 7S RNA (MT-7S), the two ribosomal RNAs MT-RNR1 (12S rRNA) and MT-RNR2 (16S rRNA), three complex IV protein mRNAs (MT-CO1, MT-CO2 and MT-CO3), three complex I protein mRNAs (MT-ND1, MT-ND5 and MT-ND6), one complex III protein mRNA (MT-CYB), and one complex V protein mRNA (MT-ATP6). Data are shown as mean ± SD, n = 4, Student’s unpaired two-tail t-test, **p
    Figure Legend Snippet: Mitochondrial transcript levels in mutant RNASEH1 fibroblasts. Mitochondrial transcript levels in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium, assessed by qPCR and normalized to GAPDH transcript levels. Analyzed transcripts included the non-coding 7S RNA (MT-7S), the two ribosomal RNAs MT-RNR1 (12S rRNA) and MT-RNR2 (16S rRNA), three complex IV protein mRNAs (MT-CO1, MT-CO2 and MT-CO3), three complex I protein mRNAs (MT-ND1, MT-ND5 and MT-ND6), one complex III protein mRNA (MT-CYB), and one complex V protein mRNA (MT-ATP6). Data are shown as mean ± SD, n = 4, Student’s unpaired two-tail t-test, **p

    Techniques Used: Mutagenesis, Real-time Polymerase Chain Reaction

    Mitochondrial translation in mutant RNASEH1 fibroblasts. (A) Western blot analysis of mitochondrial proteins involved in mitochondrial RNA metabolism (RNA metab.) and mitochondrial large (mtLSU) and small (mtSSU) ribosomal subunits in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium. GAPDH was used as loading control. GAPDH is from the same blot as Figure1D . (B) Quantification of the Western blots shown in (A) normalized to GAPDH levels. Data are shown as mean ± SD, Student’s unpaired two-tail t-test, *p
    Figure Legend Snippet: Mitochondrial translation in mutant RNASEH1 fibroblasts. (A) Western blot analysis of mitochondrial proteins involved in mitochondrial RNA metabolism (RNA metab.) and mitochondrial large (mtLSU) and small (mtSSU) ribosomal subunits in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium. GAPDH was used as loading control. GAPDH is from the same blot as Figure1D . (B) Quantification of the Western blots shown in (A) normalized to GAPDH levels. Data are shown as mean ± SD, Student’s unpaired two-tail t-test, *p

    Techniques Used: Mutagenesis, Western Blot

    Mitochondrial DNA maintenance in mutant RNASEH1 fibroblasts. (A) Western blot analysis of representative components of the mitochondrial OxPhos complexes I-V in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium. GAPDH was used as loading control. (B) Oxygen consumption ( I O2 ) measurements in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium. Values of basal and maximal respiration along with ATP production-dependent, proton leak respiration, and spare capacity are presented. Data are shown as mean ± SD, n = 4, Student’s unpaired two-tail t-test, **p
    Figure Legend Snippet: Mitochondrial DNA maintenance in mutant RNASEH1 fibroblasts. (A) Western blot analysis of representative components of the mitochondrial OxPhos complexes I-V in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium. GAPDH was used as loading control. (B) Oxygen consumption ( I O2 ) measurements in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium. Values of basal and maximal respiration along with ATP production-dependent, proton leak respiration, and spare capacity are presented. Data are shown as mean ± SD, n = 4, Student’s unpaired two-tail t-test, **p

    Techniques Used: Mutagenesis, Western Blot

    8) Product Images from "m5C modification of mRNA serves a DNA damage code to promote homologous recombination"

    Article Title: m5C modification of mRNA serves a DNA damage code to promote homologous recombination

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16722-7

    m 5 C mRNA methylation is enriched at transcriptionally active sites with DNA damage. a U2OS-TRE cells transfected with TA-KR/TA-Cherry/tetR-KR/tetR-Cherry plasmids were exposed to light for 30 min for KR activation and allowed to recover for 1 h before harvest (scale bar: 10 μm). Quantification of frequency of cells in 500 cells with m 5 C foci from three independent experiments, mean ± SD (upper right). Fold increase of m 5 C mean intensity = mean intensity of m 5 C at TA-KR/mean intensity of background ( n = 20, mean ± SD) (lower right). b U2OS-TRE cells were transfected with TA-KR/TA-Cherry to induce local oxidative damage or for the control condition. Cells were then stained for m 5 C with four different anti-m 5 C antibodies. Frequency of m 5 C-positive cells in 500 cells was quantified ( n = 3, mean ± SD). c U2OS-TRE cells transfected with TA-KR were digested with RNaseH1, RNaseA, or DNase I and stained for m 5 C quantification (scale bar: 10 μm). d The mRNA from Flp-in 293 cells treated with or without 2 mM H 2 O 2 for 40 min was used for m 5 C measurement via dot blot. Quantification of m 5 C levels (mean ± SD) from three independent experiments normalized with Ctrl and methylene blue is shown. e 32 P-labeled mRNA monophosphate nucleosides were run on 2D gels for 2D-TLC analysis. In vitro-transcribed 4B mRNA with or without m 5 C was run in parallel. Representative images from three sets of independent experiments are shown with arrows showing the directions of each solvent run. Position of each nucleotide and m 5 C are labeled (Left). f 32 P-labeled mRNA monophosphate nucleosides from U2OS cells with or without 2 mM H 2 O 2 for 40 min were run on 2D gels for 2D-TLC analysis. Representative images from three sets of independent experiments. Associated quantification of relative increase in m 5 C in peroxide-treated cells compared to control, normalized to nucleotide C (right). Statistical analysis was performed with the unpaired two tailed Student’s t -test. * p
    Figure Legend Snippet: m 5 C mRNA methylation is enriched at transcriptionally active sites with DNA damage. a U2OS-TRE cells transfected with TA-KR/TA-Cherry/tetR-KR/tetR-Cherry plasmids were exposed to light for 30 min for KR activation and allowed to recover for 1 h before harvest (scale bar: 10 μm). Quantification of frequency of cells in 500 cells with m 5 C foci from three independent experiments, mean ± SD (upper right). Fold increase of m 5 C mean intensity = mean intensity of m 5 C at TA-KR/mean intensity of background ( n = 20, mean ± SD) (lower right). b U2OS-TRE cells were transfected with TA-KR/TA-Cherry to induce local oxidative damage or for the control condition. Cells were then stained for m 5 C with four different anti-m 5 C antibodies. Frequency of m 5 C-positive cells in 500 cells was quantified ( n = 3, mean ± SD). c U2OS-TRE cells transfected with TA-KR were digested with RNaseH1, RNaseA, or DNase I and stained for m 5 C quantification (scale bar: 10 μm). d The mRNA from Flp-in 293 cells treated with or without 2 mM H 2 O 2 for 40 min was used for m 5 C measurement via dot blot. Quantification of m 5 C levels (mean ± SD) from three independent experiments normalized with Ctrl and methylene blue is shown. e 32 P-labeled mRNA monophosphate nucleosides were run on 2D gels for 2D-TLC analysis. In vitro-transcribed 4B mRNA with or without m 5 C was run in parallel. Representative images from three sets of independent experiments are shown with arrows showing the directions of each solvent run. Position of each nucleotide and m 5 C are labeled (Left). f 32 P-labeled mRNA monophosphate nucleosides from U2OS cells with or without 2 mM H 2 O 2 for 40 min were run on 2D gels for 2D-TLC analysis. Representative images from three sets of independent experiments. Associated quantification of relative increase in m 5 C in peroxide-treated cells compared to control, normalized to nucleotide C (right). Statistical analysis was performed with the unpaired two tailed Student’s t -test. * p

    Techniques Used: Methylation, Transfection, Activation Assay, Staining, Dot Blot, Labeling, Thin Layer Chromatography, In Vitro, Two Tailed Test

    9) Product Images from "Regulation of ATR activity via the RNA polymerase II associated factors CDC73 and PNUTS-PP1"

    Article Title: Regulation of ATR activity via the RNA polymerase II associated factors CDC73 and PNUTS-PP1

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1233

    PNUTS-PP1 suppresses ATR signaling. ( A ) Western blot analysis of ATR and ATM signaling events in control scrambled siRNA transfected (scr) or PNUTS siRNA transfected (siPNUTS #1 and siPNUTS #2) HeLa cells, without IR or at indicated times after 10 Gy. Cells were harvested at 72 h after siRNA transfection. Bottom bar charts show quantification of pCHK1 S317 relative to CHK1 and pRPA S33 relative to γTUBULIN levels for siPNUTS #2, hereafter called siPNUTS ( n = 8). ( B ) Western blot analysis of untreated cells or at 2 or 6 h after addition of thymidine to cells siRNA transfected as in A) (scr and siPNUTS). Bottom bar charts show quantification of pCHK1 S317 relative to CHK1 and pRPA S33 relative to RPA70 levels ( n = 10). ( C ) Western blot analysis of HeLa cells or HeLa BAC clones stably expressing EGFP mouse pnuts (mpnuts) transfected with scr or siPNUTS (specifically targets human PNUTS), without IR or at 1 or 6 h after 10 Gy. Lines to the right of the western blot indicate migration of human endogenous PNUTS (lower band) and EGFP mpnuts (upper band). Bottom bar chart shows quantification of pCHK1 S345 relative to CHK1 levels ( n = 3). ( D ) Western blot analysis of HeLa cells transfected with scr or siPNUTS. At 24 h post transfection, the indicated samples were transfected with wild type EGFP PNUTS or PP1-binding deficient EGFP PNUTS RAXA. Cells were harvested 48 h later without further treatment (–) or 1 h after 10 Gy. Lines to the right of the western blot indicate migration of endogenous PNUTS (lower band) and EGFP PNUTS/EGFP PNUTS RAXA (upper band), asterisk indicates what is likely EGFP PNUTS/EGFP PNUTS RAXA degradation products. Bar chart shows quantification of pCHK1 S317 relative to CHK1 ( n = 3). Error bars indicate standard error of the mean (SEM) and statistical significance was calculated by the two-tailed Student's two sample t-test. * P
    Figure Legend Snippet: PNUTS-PP1 suppresses ATR signaling. ( A ) Western blot analysis of ATR and ATM signaling events in control scrambled siRNA transfected (scr) or PNUTS siRNA transfected (siPNUTS #1 and siPNUTS #2) HeLa cells, without IR or at indicated times after 10 Gy. Cells were harvested at 72 h after siRNA transfection. Bottom bar charts show quantification of pCHK1 S317 relative to CHK1 and pRPA S33 relative to γTUBULIN levels for siPNUTS #2, hereafter called siPNUTS ( n = 8). ( B ) Western blot analysis of untreated cells or at 2 or 6 h after addition of thymidine to cells siRNA transfected as in A) (scr and siPNUTS). Bottom bar charts show quantification of pCHK1 S317 relative to CHK1 and pRPA S33 relative to RPA70 levels ( n = 10). ( C ) Western blot analysis of HeLa cells or HeLa BAC clones stably expressing EGFP mouse pnuts (mpnuts) transfected with scr or siPNUTS (specifically targets human PNUTS), without IR or at 1 or 6 h after 10 Gy. Lines to the right of the western blot indicate migration of human endogenous PNUTS (lower band) and EGFP mpnuts (upper band). Bottom bar chart shows quantification of pCHK1 S345 relative to CHK1 levels ( n = 3). ( D ) Western blot analysis of HeLa cells transfected with scr or siPNUTS. At 24 h post transfection, the indicated samples were transfected with wild type EGFP PNUTS or PP1-binding deficient EGFP PNUTS RAXA. Cells were harvested 48 h later without further treatment (–) or 1 h after 10 Gy. Lines to the right of the western blot indicate migration of endogenous PNUTS (lower band) and EGFP PNUTS/EGFP PNUTS RAXA (upper band), asterisk indicates what is likely EGFP PNUTS/EGFP PNUTS RAXA degradation products. Bar chart shows quantification of pCHK1 S317 relative to CHK1 ( n = 3). Error bars indicate standard error of the mean (SEM) and statistical significance was calculated by the two-tailed Student's two sample t-test. * P

    Techniques Used: Western Blot, Transfection, BAC Assay, Clone Assay, Stable Transfection, Expressing, Migration, Binding Assay, Two Tailed Test

    Depletion of PNUTS promotes R-loops, but overexpression of EGFP-RNaseH1 has only minor effects on ATR signaling. ( A ) Immunofluorescence analysis of R-loops in PNUTS depleted and control siRNA transfected cells at 72 h after siRNA transfection. The intensity of the nucleoplasmic staining is plotted. At least 50 cells from three independent experiments were scored. *** P
    Figure Legend Snippet: Depletion of PNUTS promotes R-loops, but overexpression of EGFP-RNaseH1 has only minor effects on ATR signaling. ( A ) Immunofluorescence analysis of R-loops in PNUTS depleted and control siRNA transfected cells at 72 h after siRNA transfection. The intensity of the nucleoplasmic staining is plotted. At least 50 cells from three independent experiments were scored. *** P

    Techniques Used: Over Expression, Immunofluorescence, Transfection, Staining

    CDC73, but not TOPBP1 nor ETAA1, is required for high ATR-dependent phosphorylation of both CHK1 and RPA after PNUTS depletion. ( A and B ) Western blot and quantifications ( n = 3) from cells transfected with scr, siPNUTS, and siRNA against TOPBP1 (siTOPBP1) harvested at 72 h after siRNA transfection and 1 and 6 h after 10 Gy. VE-821 was added 30 min prior to 10 Gy. For the siTOPBP1 10 Gy 6 h sample error bar was emitted in the quantifications as experiment was performed two times. Western blot for siTOPBP1 alone is shown in Supplementary Figure S5E . ( C and D ) Western blot and quantifications ( n = 3) from cells transfected with scr, siPNUTS, siTOPBP1 and siRNA against ETAA1 (siETAA1) harvested at 48 h after siRNA transfection and 1 and 6 h after 10 Gy. ( E ) Western blot analysis and quantifications of scr, siPNUTS or CDC73 siRNA (siCDC73) transfected HeLa cells or HeLa cells stably expressing siRNA-resistant Flag-CDC73 treated with IR (10Gy) as indicated. Bar charts show quantification of pCHK1 S345 and pCHK1 S317 versus CHK1 levels at 6 h after 10 Gy ( n = 3). Error bars indicate SEM and statistical significance was calculated by the two-tailed Student's two sample t -test. * P
    Figure Legend Snippet: CDC73, but not TOPBP1 nor ETAA1, is required for high ATR-dependent phosphorylation of both CHK1 and RPA after PNUTS depletion. ( A and B ) Western blot and quantifications ( n = 3) from cells transfected with scr, siPNUTS, and siRNA against TOPBP1 (siTOPBP1) harvested at 72 h after siRNA transfection and 1 and 6 h after 10 Gy. VE-821 was added 30 min prior to 10 Gy. For the siTOPBP1 10 Gy 6 h sample error bar was emitted in the quantifications as experiment was performed two times. Western blot for siTOPBP1 alone is shown in Supplementary Figure S5E . ( C and D ) Western blot and quantifications ( n = 3) from cells transfected with scr, siPNUTS, siTOPBP1 and siRNA against ETAA1 (siETAA1) harvested at 48 h after siRNA transfection and 1 and 6 h after 10 Gy. ( E ) Western blot analysis and quantifications of scr, siPNUTS or CDC73 siRNA (siCDC73) transfected HeLa cells or HeLa cells stably expressing siRNA-resistant Flag-CDC73 treated with IR (10Gy) as indicated. Bar charts show quantification of pCHK1 S345 and pCHK1 S317 versus CHK1 levels at 6 h after 10 Gy ( n = 3). Error bars indicate SEM and statistical significance was calculated by the two-tailed Student's two sample t -test. * P

    Techniques Used: Recombinase Polymerase Amplification, Western Blot, Transfection, Stable Transfection, Expressing, Two Tailed Test

    PNUTS-PP1 likely suppresses ATR signaling by dephosphorylating pRNAPII CTD. ( A ) Western blot analysis of scr or siPNUTS transfected cells without IR or 6 h after 10 Gy. VE-822 was added for 2, 5, 15, 30 or 60 min to indicated samples 6 h after 10 Gy. Charts show fold changes for VE-822-treated samples relative to the 10 Gy 6 h sample, for respective siRNA oligos from quantifications of pCHK1 S317 relative to CHK1 and pRPA S33 relative to CDK1. Experiment was performed 2 times with similar results. ( B ) Western blot analysis of scr and siPNUTS cells at 72 h after transfection. Bottom bar chart shows quantification of pRNAPII S5 relative to RNAPII ( n = 14). *** P
    Figure Legend Snippet: PNUTS-PP1 likely suppresses ATR signaling by dephosphorylating pRNAPII CTD. ( A ) Western blot analysis of scr or siPNUTS transfected cells without IR or 6 h after 10 Gy. VE-822 was added for 2, 5, 15, 30 or 60 min to indicated samples 6 h after 10 Gy. Charts show fold changes for VE-822-treated samples relative to the 10 Gy 6 h sample, for respective siRNA oligos from quantifications of pCHK1 S317 relative to CHK1 and pRPA S33 relative to CDK1. Experiment was performed 2 times with similar results. ( B ) Western blot analysis of scr and siPNUTS cells at 72 h after transfection. Bottom bar chart shows quantification of pRNAPII S5 relative to RNAPII ( n = 14). *** P

    Techniques Used: Western Blot, Transfection

    CDC73 is required for high ATR signaling in S-phase and activation of the endogenous G2 checkpoint after PNUTS depletion, and interacts with ATR and RNAPII. ( A ) Flow cytometry charts showing γH2AX staining versus DNA content as in 3A) of scr, siPNUTS or siPNUTS and siCDC73 transfected cells harvested at 72 h after siRNA transfection with and without 1 h treatment with VE-822. Quantifications show relative median γH2AX levels in indicated S-phase cells (black box). ( n = 3) * P
    Figure Legend Snippet: CDC73 is required for high ATR signaling in S-phase and activation of the endogenous G2 checkpoint after PNUTS depletion, and interacts with ATR and RNAPII. ( A ) Flow cytometry charts showing γH2AX staining versus DNA content as in 3A) of scr, siPNUTS or siPNUTS and siCDC73 transfected cells harvested at 72 h after siRNA transfection with and without 1 h treatment with VE-822. Quantifications show relative median γH2AX levels in indicated S-phase cells (black box). ( n = 3) * P

    Techniques Used: Activation Assay, Flow Cytometry, Cytometry, Staining, Transfection

    10) Product Images from "Dynamic nucleoplasmic and nucleolar localization of mammalian RNase H1 in response to RNAP I transcriptional R-loops"

    Article Title: Dynamic nucleoplasmic and nucleolar localization of mammalian RNase H1 in response to RNAP I transcriptional R-loops

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx710

    Top1 and RPA194 co-migrate to perinucleolar LNCs upon RNAP I transcriptional arrest and nucleolar segregation. ( A ) Confocal immunofluorescence imaging of HeLa cells 48 h after transfection with luciferase siRNA or Top1 siRNA. Cells were stained for Top1 and nucleolar marker NPM1. ( B ) HeLa cells treated with 250 nM CX5461 for 6 h were stained for NPM1. ( C ) Co-immunofluorescent staining of Top1 and RPA194 in HeLa cells treated with DMSO or 250 nM CX5461 (6 h).
    Figure Legend Snippet: Top1 and RPA194 co-migrate to perinucleolar LNCs upon RNAP I transcriptional arrest and nucleolar segregation. ( A ) Confocal immunofluorescence imaging of HeLa cells 48 h after transfection with luciferase siRNA or Top1 siRNA. Cells were stained for Top1 and nucleolar marker NPM1. ( B ) HeLa cells treated with 250 nM CX5461 for 6 h were stained for NPM1. ( C ) Co-immunofluorescent staining of Top1 and RPA194 in HeLa cells treated with DMSO or 250 nM CX5461 (6 h).

    Techniques Used: Immunofluorescence, Imaging, Transfection, Luciferase, Staining, Marker

    11) Product Images from "RNase H1 Regulates Mitochondrial Transcription and Translation via the Degradation of 7S RNA"

    Article Title: RNase H1 Regulates Mitochondrial Transcription and Translation via the Degradation of 7S RNA

    Journal: Frontiers in Genetics

    doi: 10.3389/fgene.2019.01393

    Mitochondrial DNA maintenance in mutant RNASEH1 fibroblasts. (A) Southern blot of total DNA digested with Pvu II from control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium. A radioactive probe against mtDNA was used to detect both linearized mtDNA (empty arrowhead) and 7S DNA (filled arrowhead and bracket), while a probe against 18S rDNA was used as loading control. (B) Relative mitochondrial DNA copy number in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium, calculated as the linearized mtDNA/18S rDNA signal ratio. Data are shown as mean ± SD, n = 4, ***p
    Figure Legend Snippet: Mitochondrial DNA maintenance in mutant RNASEH1 fibroblasts. (A) Southern blot of total DNA digested with Pvu II from control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium. A radioactive probe against mtDNA was used to detect both linearized mtDNA (empty arrowhead) and 7S DNA (filled arrowhead and bracket), while a probe against 18S rDNA was used as loading control. (B) Relative mitochondrial DNA copy number in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium, calculated as the linearized mtDNA/18S rDNA signal ratio. Data are shown as mean ± SD, n = 4, ***p

    Techniques Used: Mutagenesis, Southern Blot

    RNASEH1 mutations, transcript, and protein levels. (A) Domains ofhuman RNAse H1 protein (MTS, mitochondrial targeting sequence; HBD, hybrid binding domain; CD, connection domain; catalytic domain). RNase H1 protein sequences from representative species, H. sapiens (Hs, NP_002927) M. musculus (Mm, NP_035405), B. taurus (Bt, NP_001039970), G. gallus (Gg, NP_990329), X. tropicalis (Xt, NP_001096299), D. rerio (Dr, NP_001002659), C. intestinalis (Ci, F6QPH0), D. melanogaster (Dm, NP_995777), C. elegans (Ce, NP_001040786), S. cerevisiae (Sc, Q04740), were extracted from the database and aligned using ClustalW2. Conserved residues found mutated in the patient in exon 4 are boxed in red, while residues in the active site and interacting with DNA or RNA are boxed in yellow and green, respectively. Positions of β strands are marked by blue arrows. (B) Human RNase H1 crystal structure (PDB ID 2QK9) 18 bp DNA(cyan): RNA (magenta) hybrid is shown respectively. Residues in the active site are colored in yellow. Residues Trp 164 (green) and Val 142 , or the mutated variant Ile 142 (red), are shown as sticks. (C) RNASEH1 transcript levels in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium assessed by qPCR (probe Hs00268000_m1) and normalized to GAPDH transcript levels. Data are shown as mean ± SD, n = 4, ***p
    Figure Legend Snippet: RNASEH1 mutations, transcript, and protein levels. (A) Domains ofhuman RNAse H1 protein (MTS, mitochondrial targeting sequence; HBD, hybrid binding domain; CD, connection domain; catalytic domain). RNase H1 protein sequences from representative species, H. sapiens (Hs, NP_002927) M. musculus (Mm, NP_035405), B. taurus (Bt, NP_001039970), G. gallus (Gg, NP_990329), X. tropicalis (Xt, NP_001096299), D. rerio (Dr, NP_001002659), C. intestinalis (Ci, F6QPH0), D. melanogaster (Dm, NP_995777), C. elegans (Ce, NP_001040786), S. cerevisiae (Sc, Q04740), were extracted from the database and aligned using ClustalW2. Conserved residues found mutated in the patient in exon 4 are boxed in red, while residues in the active site and interacting with DNA or RNA are boxed in yellow and green, respectively. Positions of β strands are marked by blue arrows. (B) Human RNase H1 crystal structure (PDB ID 2QK9) 18 bp DNA(cyan): RNA (magenta) hybrid is shown respectively. Residues in the active site are colored in yellow. Residues Trp 164 (green) and Val 142 , or the mutated variant Ile 142 (red), are shown as sticks. (C) RNASEH1 transcript levels in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium assessed by qPCR (probe Hs00268000_m1) and normalized to GAPDH transcript levels. Data are shown as mean ± SD, n = 4, ***p

    Techniques Used: Sequencing, Binding Assay, Variant Assay, Real-time Polymerase Chain Reaction

    Mitochondrial transcript levels in mutant RNASEH1 fibroblasts. Mitochondrial transcript levels in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium, assessed by qPCR and normalized to GAPDH transcript levels. Analyzed transcripts included the non-coding 7S RNA (MT-7S), the two ribosomal RNAs MT-RNR1 (12S rRNA) and MT-RNR2 (16S rRNA), three complex IV protein mRNAs (MT-CO1, MT-CO2 and MT-CO3), three complex I protein mRNAs (MT-ND1, MT-ND5 and MT-ND6), one complex III protein mRNA (MT-CYB), and one complex V protein mRNA (MT-ATP6). Data are shown as mean ± SD, n = 4, Student’s unpaired two-tail t-test, **p
    Figure Legend Snippet: Mitochondrial transcript levels in mutant RNASEH1 fibroblasts. Mitochondrial transcript levels in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium, assessed by qPCR and normalized to GAPDH transcript levels. Analyzed transcripts included the non-coding 7S RNA (MT-7S), the two ribosomal RNAs MT-RNR1 (12S rRNA) and MT-RNR2 (16S rRNA), three complex IV protein mRNAs (MT-CO1, MT-CO2 and MT-CO3), three complex I protein mRNAs (MT-ND1, MT-ND5 and MT-ND6), one complex III protein mRNA (MT-CYB), and one complex V protein mRNA (MT-ATP6). Data are shown as mean ± SD, n = 4, Student’s unpaired two-tail t-test, **p

    Techniques Used: Mutagenesis, Real-time Polymerase Chain Reaction

    Mitochondrial translation in mutant RNASEH1 fibroblasts. (A) Western blot analysis of mitochondrial proteins involved in mitochondrial RNA metabolism (RNA metab.) and mitochondrial large (mtLSU) and small (mtSSU) ribosomal subunits in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium. GAPDH was used as loading control. GAPDH is from the same blot as Figure1D . (B) Quantification of the Western blots shown in (A) normalized to GAPDH levels. Data are shown as mean ± SD, Student’s unpaired two-tail t-test, *p
    Figure Legend Snippet: Mitochondrial translation in mutant RNASEH1 fibroblasts. (A) Western blot analysis of mitochondrial proteins involved in mitochondrial RNA metabolism (RNA metab.) and mitochondrial large (mtLSU) and small (mtSSU) ribosomal subunits in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium. GAPDH was used as loading control. GAPDH is from the same blot as Figure1D . (B) Quantification of the Western blots shown in (A) normalized to GAPDH levels. Data are shown as mean ± SD, Student’s unpaired two-tail t-test, *p

    Techniques Used: Mutagenesis, Western Blot

    Mitochondrial DNA maintenance in mutant RNASEH1 fibroblasts. (A) Western blot analysis of representative components of the mitochondrial OxPhos complexes I-V in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium. GAPDH was used as loading control. (B) Oxygen consumption ( I O2 ) measurements in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium. Values of basal and maximal respiration along with ATP production-dependent, proton leak respiration, and spare capacity are presented. Data are shown as mean ± SD, n = 4, Student’s unpaired two-tail t-test, **p
    Figure Legend Snippet: Mitochondrial DNA maintenance in mutant RNASEH1 fibroblasts. (A) Western blot analysis of representative components of the mitochondrial OxPhos complexes I-V in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium. GAPDH was used as loading control. (B) Oxygen consumption ( I O2 ) measurements in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium. Values of basal and maximal respiration along with ATP production-dependent, proton leak respiration, and spare capacity are presented. Data are shown as mean ± SD, n = 4, Student’s unpaired two-tail t-test, **p

    Techniques Used: Mutagenesis, Western Blot

    12) Product Images from "BRCA2 controls DNA:RNA hybrid level at DSBs by mediating RNase H2 recruitment"

    Article Title: BRCA2 controls DNA:RNA hybrid level at DSBs by mediating RNase H2 recruitment

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07799-2

    DNA:RNA hybrids are directly recognized by BRCA1 in vitro and in vivo. a Representative pictures of super-resolution imaging analysis of BRCA1 (cyan) and DNA:RNA hybrids (yellow) colocalization in S-phase synchronized NCS-treated U2OS cells. Scale bar: 5 μm. b Dot plot shows the normalized number of overlaps relative to random of BRCA1 and DNA:RNA hybrids signals in S-phase U2OS cells treated with DSMO or NCS. At least n = 40 events were counted from three independent experiments. Lines represent mean ± s.e.m. c Electrophoretic mobility shift assay (EMSA) of purified recombinant human BRCA1 or BRCA1-BARD1 with end-labeled (*) double-stranded DNA or DNA:RNA substrates. d Graph showing the percentage of protein-bound substrate at respective protein concentrations. Error bars represent s.e.m. ( n = 2 independent experiments). e Representative images of BRCA1 foci co-stained with cyclin A, as S/G2-phase marker, in irradiated (2 Gy) U2OS cells over-expressing GFP or GFP-RNase H1 (GFP-RH1). Scale bar: 5 μm. f Dot plot shows the number of foci in e . At least n = 80 cells were counted from at least three independent experiments. Lines represent mean ± s.e.m. g Representative images of BRCA1 foci co-stained with cyclin A, as S/G2-phase marker, in irradiated (2 Gy) U2OS cells treated with RNase H prior to fixation. Scale bar: 10 μm. h Dot plot shows the number of foci in g . At least n = 80 cells were counted from three independent experiments. Lines represent mean ± s.e.m. * P
    Figure Legend Snippet: DNA:RNA hybrids are directly recognized by BRCA1 in vitro and in vivo. a Representative pictures of super-resolution imaging analysis of BRCA1 (cyan) and DNA:RNA hybrids (yellow) colocalization in S-phase synchronized NCS-treated U2OS cells. Scale bar: 5 μm. b Dot plot shows the normalized number of overlaps relative to random of BRCA1 and DNA:RNA hybrids signals in S-phase U2OS cells treated with DSMO or NCS. At least n = 40 events were counted from three independent experiments. Lines represent mean ± s.e.m. c Electrophoretic mobility shift assay (EMSA) of purified recombinant human BRCA1 or BRCA1-BARD1 with end-labeled (*) double-stranded DNA or DNA:RNA substrates. d Graph showing the percentage of protein-bound substrate at respective protein concentrations. Error bars represent s.e.m. ( n = 2 independent experiments). e Representative images of BRCA1 foci co-stained with cyclin A, as S/G2-phase marker, in irradiated (2 Gy) U2OS cells over-expressing GFP or GFP-RNase H1 (GFP-RH1). Scale bar: 5 μm. f Dot plot shows the number of foci in e . At least n = 80 cells were counted from at least three independent experiments. Lines represent mean ± s.e.m. g Representative images of BRCA1 foci co-stained with cyclin A, as S/G2-phase marker, in irradiated (2 Gy) U2OS cells treated with RNase H prior to fixation. Scale bar: 10 μm. h Dot plot shows the number of foci in g . At least n = 80 cells were counted from three independent experiments. Lines represent mean ± s.e.m. * P

    Techniques Used: In Vitro, In Vivo, Imaging, Electrophoretic Mobility Shift Assay, Purification, Recombinant, Labeling, Staining, Marker, Irradiation, Expressing

    13) Product Images from "RNase H1 promotes replication fork progression through oppositely transcribed regions of Drosophila mitochondrial DNA"

    Article Title: RNase H1 promotes replication fork progression through oppositely transcribed regions of Drosophila mitochondrial DNA

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.007015

    Overexpression of rnh1 relieves replication pausing. A–D , 2DNAGE of four restriction fragments of Drosophila S2 cells mtDNA, probed as indicated, in material from control cells and cells overexpressing RNase H1 in the form of epitope-tagged RNase H1-V5 (denoted OE ), both treated with 500 μ m CuSO 4 for 48 h to induce expression. E , schematic map of Drosophila mtDNA, as also shown in Fig. 8 , indicating the location of relevant restriction sites ( open circles ), mTTF-binding sites (bs1 and bs2; filled circles ), the noncoding region ( bold ), and the probes used. The open arrowhead marks the location and direction of replication initiation (see Ref. 40 ). The directions of first- and second-dimension electrophoresis in all gels are as indicated by the arrows . The images show relatively low exposures to reveal fine details of the arcs of RIs.
    Figure Legend Snippet: Overexpression of rnh1 relieves replication pausing. A–D , 2DNAGE of four restriction fragments of Drosophila S2 cells mtDNA, probed as indicated, in material from control cells and cells overexpressing RNase H1 in the form of epitope-tagged RNase H1-V5 (denoted OE ), both treated with 500 μ m CuSO 4 for 48 h to induce expression. E , schematic map of Drosophila mtDNA, as also shown in Fig. 8 , indicating the location of relevant restriction sites ( open circles ), mTTF-binding sites (bs1 and bs2; filled circles ), the noncoding region ( bold ), and the probes used. The open arrowhead marks the location and direction of replication initiation (see Ref. 40 ). The directions of first- and second-dimension electrophoresis in all gels are as indicated by the arrows . The images show relatively low exposures to reveal fine details of the arcs of RIs.

    Techniques Used: Over Expression, Expressing, Binding Assay, Electrophoresis

    Subcellular localization of epitope-tagged RNase H1. A , immunocytochemistry of cells transiently transfected with RNase H1-V5, probed for the V5 epitope tag ( red ), Cox4 ( green ), and DAPI ( blue ), showing examples of the three types of intracellular distribution of V5-tagged RNase H1: nucleus and mitochondria ( i ), mitochondria only ( ii ), and nucleus only ( iii ). B , subcellular distribution of RNase H1-V5 in 100 transfected cells as indicated (mean of three experiments, error bars denote S.D.). C , Western blots of subcellular fractions from cells transfected with RNase H1-V5, highly enriched for nuclei ( nuc ) or mitochondria ( mt ) as indicated, probed simultaneously for V5 and for the markers indicated. M , molecular mass markers.
    Figure Legend Snippet: Subcellular localization of epitope-tagged RNase H1. A , immunocytochemistry of cells transiently transfected with RNase H1-V5, probed for the V5 epitope tag ( red ), Cox4 ( green ), and DAPI ( blue ), showing examples of the three types of intracellular distribution of V5-tagged RNase H1: nucleus and mitochondria ( i ), mitochondria only ( ii ), and nucleus only ( iii ). B , subcellular distribution of RNase H1-V5 in 100 transfected cells as indicated (mean of three experiments, error bars denote S.D.). C , Western blots of subcellular fractions from cells transfected with RNase H1-V5, highly enriched for nuclei ( nuc ) or mitochondria ( mt ) as indicated, probed simultaneously for V5 and for the markers indicated. M , molecular mass markers.

    Techniques Used: Immunocytochemistry, Transfection, Western Blot

    Subcellular targeting of RNase H1 variants. A , intracellular localization of RNase H1-V5 variants in cultures of stably transfected cells exemplified in B . M1V and M16V, N-terminal methionine variants (see Fig. S2 A ); ΔNLS, with the putative nuclear localization signal deleted (see Fig. S2 C ). C , intracellular localization of RNase H1-V5 in cells synchronized in G1 and G2 (see FACS profiles in Fig. S2 E ). All plotted values are means of three experiments. Error bars denote S.D. nuc , nuclei; mt , mitochondria.
    Figure Legend Snippet: Subcellular targeting of RNase H1 variants. A , intracellular localization of RNase H1-V5 variants in cultures of stably transfected cells exemplified in B . M1V and M16V, N-terminal methionine variants (see Fig. S2 A ); ΔNLS, with the putative nuclear localization signal deleted (see Fig. S2 C ). C , intracellular localization of RNase H1-V5 in cells synchronized in G1 and G2 (see FACS profiles in Fig. S2 E ). All plotted values are means of three experiments. Error bars denote S.D. nuc , nuclei; mt , mitochondria.

    Techniques Used: Stable Transfection, Transfection, FACS

    14) Product Images from "Human RNase H1 Is Associated with Protein P32 and Is Involved in Mitochondrial Pre-rRNA Processing"

    Article Title: Human RNase H1 Is Associated with Protein P32 and Is Involved in Mitochondrial Pre-rRNA Processing

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0071006

    Both RNase H1 and P32 interact with mitochondrial DNA and pre-rRNA. ( A ) The positions of Probes and PCR primers for the human mitochondrial DNA. The DNA map was derived from published review [65] . Two oligonucleotide probes specific to 12 S and 16 S mitochondria rRNA regions are shown in Blue bars . Three sets of PCR probes corresponding to the A, B and C regions are indicated in Green arrows . ( B ) RNase H1 and P32 bind mitochondrial DNA. Cell extracts were prepared from an HA-H1 stably expressing cell line (RNase H1), control HEK cells or HEK cells transfected with the HA-P32 expression plasmid (P32). Equal amounts of each extract were used for immunoprecipitation with anti-HA beads. Nucleic acids were extracted from the precipitated samples using phenol/chloroform and subjected to PCR analysis. The probe sets for PCR were shown in Figure 6A . Genomic DNA from HEK cells that was used as a positive control. The PCR products were analyzed on 2% Agarose gels. ( C ) RNase H1 may interact with the mitochondrial rDNA region. The extracts from HA-H1 cell and control HEK cells were used for immunoprecipitation with HA-antibody. The precipitates were digested on beads with (+) or without (−) DNase I. The DNA associated with beads was then extracted and subjected to PCR analysis. The PCR products were separated in 2% agarose gel. ( D ) RNase H1 and P32 also co-immunoprecipitated with mitochondrial pre-rRNA. The same extracted nucleic acids from panel B were digested with DNase I. The RNA is used for reverse transcription with (+) or without (−) reverse transcriptase, followed by PCR amplification using different primer sets as indicated below the panels. PCR reaction using primers specific to U16 snoRNA was used as control.
    Figure Legend Snippet: Both RNase H1 and P32 interact with mitochondrial DNA and pre-rRNA. ( A ) The positions of Probes and PCR primers for the human mitochondrial DNA. The DNA map was derived from published review [65] . Two oligonucleotide probes specific to 12 S and 16 S mitochondria rRNA regions are shown in Blue bars . Three sets of PCR probes corresponding to the A, B and C regions are indicated in Green arrows . ( B ) RNase H1 and P32 bind mitochondrial DNA. Cell extracts were prepared from an HA-H1 stably expressing cell line (RNase H1), control HEK cells or HEK cells transfected with the HA-P32 expression plasmid (P32). Equal amounts of each extract were used for immunoprecipitation with anti-HA beads. Nucleic acids were extracted from the precipitated samples using phenol/chloroform and subjected to PCR analysis. The probe sets for PCR were shown in Figure 6A . Genomic DNA from HEK cells that was used as a positive control. The PCR products were analyzed on 2% Agarose gels. ( C ) RNase H1 may interact with the mitochondrial rDNA region. The extracts from HA-H1 cell and control HEK cells were used for immunoprecipitation with HA-antibody. The precipitates were digested on beads with (+) or without (−) DNase I. The DNA associated with beads was then extracted and subjected to PCR analysis. The PCR products were separated in 2% agarose gel. ( D ) RNase H1 and P32 also co-immunoprecipitated with mitochondrial pre-rRNA. The same extracted nucleic acids from panel B were digested with DNase I. The RNA is used for reverse transcription with (+) or without (−) reverse transcriptase, followed by PCR amplification using different primer sets as indicated below the panels. PCR reaction using primers specific to U16 snoRNA was used as control.

    Techniques Used: Polymerase Chain Reaction, Derivative Assay, Stable Transfection, Expressing, Transfection, Plasmid Preparation, Immunoprecipitation, Positive Control, Agarose Gel Electrophoresis, Amplification

    Depletion of RNase H1 or P32 resulted in accumulation of mitochondrial pre-12S/16S rRNA. HeLa cells were treated with 2 nM or 20 nM of RNase H1-siRNA or P32 –siRNA for 24 or 48 hours. ( A ) The mRNA levels of RNase H1 and P32 were determined by qRT-PCR 24 hrs after siRNA treatment. ( B ) Protein levels of RNase H1 and P32 were analyzed by western analysis 24 hours post siRNA treatment. ( C ) Reduction of RNase H1 or P32 significantly increased the level of mitochondrial pre-rRNA. HeLa cells were treated with either RNase H1-siRNA (2 nM) or P32-siRNA (2 nM) for 24 hours. Total RNA was prepared and subjected to Northern analysis with 32 P labeled probes specific to 12S or 16S rRNAs. U3 snoRNA was detected and served as a control. The relative levels of pre-rRNA were measured from the results obtained with 12 S probe, normalized to U3, and plotted in the right panel. The error bars indicate standard error of the three replicates. (D) RT-PCR assay for the levels of pre-16 S and pre-ND3 RNAs. Total RNA prepared from HeLa cells treated for 24 hrs with corresponding siRNAs was analyzed by qRT-PCR, using primer probe sets specific to the tRNA Val -16 S rRNA junction (pre-16 S) or to the tRNA Gly -ND3 junction (pre-ND3). The error bars represent standard deviation of three replicates.
    Figure Legend Snippet: Depletion of RNase H1 or P32 resulted in accumulation of mitochondrial pre-12S/16S rRNA. HeLa cells were treated with 2 nM or 20 nM of RNase H1-siRNA or P32 –siRNA for 24 or 48 hours. ( A ) The mRNA levels of RNase H1 and P32 were determined by qRT-PCR 24 hrs after siRNA treatment. ( B ) Protein levels of RNase H1 and P32 were analyzed by western analysis 24 hours post siRNA treatment. ( C ) Reduction of RNase H1 or P32 significantly increased the level of mitochondrial pre-rRNA. HeLa cells were treated with either RNase H1-siRNA (2 nM) or P32-siRNA (2 nM) for 24 hours. Total RNA was prepared and subjected to Northern analysis with 32 P labeled probes specific to 12S or 16S rRNAs. U3 snoRNA was detected and served as a control. The relative levels of pre-rRNA were measured from the results obtained with 12 S probe, normalized to U3, and plotted in the right panel. The error bars indicate standard error of the three replicates. (D) RT-PCR assay for the levels of pre-16 S and pre-ND3 RNAs. Total RNA prepared from HeLa cells treated for 24 hrs with corresponding siRNAs was analyzed by qRT-PCR, using primer probe sets specific to the tRNA Val -16 S rRNA junction (pre-16 S) or to the tRNA Gly -ND3 junction (pre-ND3). The error bars represent standard deviation of three replicates.

    Techniques Used: Quantitative RT-PCR, Western Blot, Northern Blot, Labeling, Reverse Transcription Polymerase Chain Reaction, Standard Deviation

    Co-localization of P32 and RNase H1. ( A ) Immunofluorescence Staining of P32 and RNase H1. Upper panel: HeLa cells were stained for endogenous P32 and RNase H1 using mouse monoclonal anti-P32 antibody and rabbit anti-RNase H1 antibody, respectively, followed by FITC conjugated donkey anti-mouse ( green ) and TRITC conjugated anti-rabbit secondary antibodies ( red ). Nuclei were stained with DAP1 ( Blue ) and Mitochondria were stained with mitotracker ( white ). Lower panel: HeLa cells were infected with adenovirus expressing RNase H1. Cells were stained as described in upper panel. ( B ) Subcellular fractionation of P32 protein. The proteins from sub-cellular compartments (cytosol, mitochondrial and ER membranes, nucleus and cytoskeleton) were prepared from HEK cells using proteome cell compartment kit (Qiagen). About 10 µg protein samples from each fraction were analyzed by western for P32. The same blot was stripped and tubulin-γ was detected to serve as a control.
    Figure Legend Snippet: Co-localization of P32 and RNase H1. ( A ) Immunofluorescence Staining of P32 and RNase H1. Upper panel: HeLa cells were stained for endogenous P32 and RNase H1 using mouse monoclonal anti-P32 antibody and rabbit anti-RNase H1 antibody, respectively, followed by FITC conjugated donkey anti-mouse ( green ) and TRITC conjugated anti-rabbit secondary antibodies ( red ). Nuclei were stained with DAP1 ( Blue ) and Mitochondria were stained with mitotracker ( white ). Lower panel: HeLa cells were infected with adenovirus expressing RNase H1. Cells were stained as described in upper panel. ( B ) Subcellular fractionation of P32 protein. The proteins from sub-cellular compartments (cytosol, mitochondrial and ER membranes, nucleus and cytoskeleton) were prepared from HEK cells using proteome cell compartment kit (Qiagen). About 10 µg protein samples from each fraction were analyzed by western for P32. The same blot was stripped and tubulin-γ was detected to serve as a control.

    Techniques Used: Immunofluorescence, Staining, Infection, Expressing, Fractionation, Western Blot

    Recombinant P32 binds to recombinant RNase H1, enhances its turnover rate, and reduces the binding affinity of the enzyme for the heteroduplex substrate. ( A ) Coomassie blue staining of the purified human His-H1, GST protein, and GST-P32 proteins separated by SDS-PAGE. The sizes for the standard protein markers are indicated. ( B ) RNase H1 but not P32 appears to bind the heteroduplex substrate. Gel shift assay was performed using 0.4 ug purified RNase H1, GST-P32, or GST proteins incubated at 4°C for 30 min with a non-cleavable heteroduplex containing 32 P labeled uniformly modified 2′-fluoro RNA annealed to DNA and subjected to native gel electrophoresis. ( C ) The interaction between RNase H1 and P32 appears to be equal molar. A fixed amount of GST-P32 was bound to GST affinity beads and then incubated with increasing amounts of RNase H1. Glutathione (GSH) eluted RNase H1 and P32 were quantified by Western blot as described in the Material and Methods. The amounts of bead-bound P32 and P32-associated RNase H1 were determined by loading known amounts of the respective proteins (left panel). The molecular ratio of bound RNase H1 relative to P32 was calculated and plotted in the right panel. ( D ) The effects of ionic strength on RNase H1/P32 interaction. Left panel: RNase H1 binds GST-P32 but not GST protein. GST or GST-P32 bound to anti-GST beads was incubated with RNase H1 in NaCl concentrations ranging from 0-950 mM as described in the Material and Methods. Middle panel: increasing NaCl concentration inhibits binding of RNase H1 to P32. Both unbound (flow through) and bound (affinity eluted) fractions were collected and the levels of RNase H1 and P32 evaluated by western blot. Right panel: Increasing pH reduced binding of RNase H1 to P32. ( E ) Michaelis-Menten kinetics and binding constants for RNase H1 cleavage of an RNA/DNA duplex in the presence or absence of P32. The K m , V max , and K d were determined by incubating the Apo B RNA/DNA duplex with RNase H1 plus GST (as control) or RNase H1 plus different amounts of P32 resulting in an H1:P32 ratio = 1∶1 or 1∶5. An uncleavable competitive inhibitor (2′-fluororibonucleotide/DNA) was used to determine the binding to the RNA/DNA duplex, as described in the Material and Methods. The calculated constants are indicated in the right panel. The error bars indicate the standard error from three parallel experiments.
    Figure Legend Snippet: Recombinant P32 binds to recombinant RNase H1, enhances its turnover rate, and reduces the binding affinity of the enzyme for the heteroduplex substrate. ( A ) Coomassie blue staining of the purified human His-H1, GST protein, and GST-P32 proteins separated by SDS-PAGE. The sizes for the standard protein markers are indicated. ( B ) RNase H1 but not P32 appears to bind the heteroduplex substrate. Gel shift assay was performed using 0.4 ug purified RNase H1, GST-P32, or GST proteins incubated at 4°C for 30 min with a non-cleavable heteroduplex containing 32 P labeled uniformly modified 2′-fluoro RNA annealed to DNA and subjected to native gel electrophoresis. ( C ) The interaction between RNase H1 and P32 appears to be equal molar. A fixed amount of GST-P32 was bound to GST affinity beads and then incubated with increasing amounts of RNase H1. Glutathione (GSH) eluted RNase H1 and P32 were quantified by Western blot as described in the Material and Methods. The amounts of bead-bound P32 and P32-associated RNase H1 were determined by loading known amounts of the respective proteins (left panel). The molecular ratio of bound RNase H1 relative to P32 was calculated and plotted in the right panel. ( D ) The effects of ionic strength on RNase H1/P32 interaction. Left panel: RNase H1 binds GST-P32 but not GST protein. GST or GST-P32 bound to anti-GST beads was incubated with RNase H1 in NaCl concentrations ranging from 0-950 mM as described in the Material and Methods. Middle panel: increasing NaCl concentration inhibits binding of RNase H1 to P32. Both unbound (flow through) and bound (affinity eluted) fractions were collected and the levels of RNase H1 and P32 evaluated by western blot. Right panel: Increasing pH reduced binding of RNase H1 to P32. ( E ) Michaelis-Menten kinetics and binding constants for RNase H1 cleavage of an RNA/DNA duplex in the presence or absence of P32. The K m , V max , and K d were determined by incubating the Apo B RNA/DNA duplex with RNase H1 plus GST (as control) or RNase H1 plus different amounts of P32 resulting in an H1:P32 ratio = 1∶1 or 1∶5. An uncleavable competitive inhibitor (2′-fluororibonucleotide/DNA) was used to determine the binding to the RNA/DNA duplex, as described in the Material and Methods. The calculated constants are indicated in the right panel. The error bars indicate the standard error from three parallel experiments.

    Techniques Used: Recombinant, Binding Assay, Staining, Purification, SDS Page, Electrophoretic Mobility Shift Assay, Incubation, Labeling, Modification, Nucleic Acid Electrophoresis, Western Blot, Concentration Assay, Flow Cytometry

    P32 appears to interact with the N-terminal duplex binding domain of RNase H1. ( A ) Expression and purification of RNase H1 deletion mutants. Left panel: Schematic depiction of the different human RNase H1 deletion mutants. DL1 deletes the hybrid binding domain (amino acid positions 1–73); DL2 deletes both the hybrid binding domain and the spacer domain (amino acid 1–129). The black bars at the N-terminus of each mutant represent a His tag. Right panel: Coomassie blue staining of the purified RNase H1 deletion mutants. The sizes of the standard markers are given. ( B ) Interaction of full length RNase H1 and its deletion mutants with P32. The full length or truncated RNase H1 proteins were incubated with GST-P32 bound to GST-beads under different NaCl concentrations ranging from 150–450 mM in both the binding and washing solutions. The P32 and RNase H1 or deletion mutants were eluted and analyzed by Western blot, using P32 or RNase H1 antibodies, respectively (right panel). Western blot to RNase H1 and deletion mutants DL1 and DL2 demonstrates that the mutant proteins are recognized by the RNase H1 antibody (left panel). ( C ) Michaelis-Menten Kinetics of DL-1 mutant in the presence or absence of P32. K m , V max , and k cat for DL-1 plus GST or GST-P32 (DL-1:P32 = 1:5 in molecular ratio) were determined in 50 and 150 mM NaCl concentration with the Apo B RNA/DNA duplex as described in the Material and Methods.
    Figure Legend Snippet: P32 appears to interact with the N-terminal duplex binding domain of RNase H1. ( A ) Expression and purification of RNase H1 deletion mutants. Left panel: Schematic depiction of the different human RNase H1 deletion mutants. DL1 deletes the hybrid binding domain (amino acid positions 1–73); DL2 deletes both the hybrid binding domain and the spacer domain (amino acid 1–129). The black bars at the N-terminus of each mutant represent a His tag. Right panel: Coomassie blue staining of the purified RNase H1 deletion mutants. The sizes of the standard markers are given. ( B ) Interaction of full length RNase H1 and its deletion mutants with P32. The full length or truncated RNase H1 proteins were incubated with GST-P32 bound to GST-beads under different NaCl concentrations ranging from 150–450 mM in both the binding and washing solutions. The P32 and RNase H1 or deletion mutants were eluted and analyzed by Western blot, using P32 or RNase H1 antibodies, respectively (right panel). Western blot to RNase H1 and deletion mutants DL1 and DL2 demonstrates that the mutant proteins are recognized by the RNase H1 antibody (left panel). ( C ) Michaelis-Menten Kinetics of DL-1 mutant in the presence or absence of P32. K m , V max , and k cat for DL-1 plus GST or GST-P32 (DL-1:P32 = 1:5 in molecular ratio) were determined in 50 and 150 mM NaCl concentration with the Apo B RNA/DNA duplex as described in the Material and Methods.

    Techniques Used: Binding Assay, Expressing, Purification, Mutagenesis, Staining, Incubation, Western Blot, Concentration Assay

    Human RNase H1 is associated with P32. ( A ) Western blot analysis of cell lysates and immunoprecipitated samples show Flag-tagged RNase H1 and H2 expression from cells stably transformed with RNase H1 (H1) or H2 (H2) or wild type (control) HEK cell lines. ( B ) Co-selection of RNase H1 binding proteins by immunoprecipitation. Extracts from cells expressing the Flag-H1, Flag-H2, or HA-H1 cell lines were immunoprecipitated with either anti-Flag or anti-HA antibody. Co-precipitated proteins were resolved by SDS-PAGE, and visualized by silver staining. Protein bands that were different from the co-precipitated proteins from control cells were subjected to mass spectrometry. The protein bands corresponding to the tagged RNase H1, H2 and the co-precipitated P32 proteins are indicated. The size marker was SeeBlue Plus2 Pre-Stained Standard (Invitrogen). ( C ) 2D gel electrophoresis of proteins co-precipitated with Flag-H1 or Flag-H2. About 5 mg cell lysates were prepared for immunoprecipitation with anti-flag beads from cell lines which stably express Flag-H1 or Flag-H2. The immunoprecipitates were washed four times with RIPA buffer and directly sent to Applied Biomics Inc. (San Francisco, CA) for 2D gel electrophoresis coupled with MS analysis. In brief, the co-precipitated proteins from Flag-H1 or Flag-H2 cells were labeled by fluorescent DIGE CyDyers, respectively, followed by 2D gel electrophoresis. The protein image was scanned with a fluorescence detector. The figure illustrates the proteins differentially associated with RNase H1 (green) or H2 (red). The P32 protein was confirmed with mass spectrum from the extracted gel sample. Circled spots were identified as RNase H1, H2 or P32 by mass spectrometric analysis. ( D ) Both endogenous and expressed RNase H1 are co-precipitated with the expressed P32. Left panel: western blots with P32, RNase H1, or H2 antibodies for proteins co-precipitated using anti-HA antibody from extracts of control HeLa cells or cells transfected with HA-P32 expression plasmid. Right panel: western blots for proteins co-selected using anti-HA antibody from extracts of Flag-H1, Flag-H2 stable cell lines and control cells, all of which were transfected with HA-P32 expression plasmid. ( E ) Confirmation of the specific interaction between RNase H1 and P32. RNase H cleavage activity indicates that the P32 co-immunoprecipitated material contains only RNase H1 enzyme activity. Upper panel: Cleavage patterns of human RNase H1 and H2 from IP-coupled enzyme activity assays. Immunoprecipitations were performed with either anti-flag, anti-RNase H1 or anti-H2 antibodies from extracts of Flag-H1, Flag-H2 expressing cells or control cells. The co-precipitated samples were incubated for the indicated times with a 32 P-labeled RNA/DNA-methoxyethyl (MOE) gapmer duplex and the cleavage products were separated using denaturing gel electrophoresis. The preferred cleavage sites of RNase H1 and H2 are indicated with * or #, respectively. The positions of the preferred cleavage sites in the heteroduplex are shown in the middle panel with the sequences of the RNA substrate (upper strand) and the oligonucleotide (lower strand). The bold nucleotides in the oligonucleotide strand indicate the position of the MOE substitutions. Lower panel: only the RNase H1 enzyme activity was detected in the co-precipitated material from lysates containing tagged P32. Immunoprecipitations were performed with anti-HA antibody from extracts of Flag-H1 or Flag-H2 stable cell lines or control HEK cells, which were all transfected or not transfected with HA-P32 expression plasmid. The precipitated samples were analyzed for cleavage patterns as described above. The position of the cleavage bands relative to the sequence of the cleavage products is shown on the left. A partial alkaline digestion of the same labeled RNA was used as a sequence ladder. The cleavage pattern of purified human RNase H1 is shown at the far right of the lower panel.
    Figure Legend Snippet: Human RNase H1 is associated with P32. ( A ) Western blot analysis of cell lysates and immunoprecipitated samples show Flag-tagged RNase H1 and H2 expression from cells stably transformed with RNase H1 (H1) or H2 (H2) or wild type (control) HEK cell lines. ( B ) Co-selection of RNase H1 binding proteins by immunoprecipitation. Extracts from cells expressing the Flag-H1, Flag-H2, or HA-H1 cell lines were immunoprecipitated with either anti-Flag or anti-HA antibody. Co-precipitated proteins were resolved by SDS-PAGE, and visualized by silver staining. Protein bands that were different from the co-precipitated proteins from control cells were subjected to mass spectrometry. The protein bands corresponding to the tagged RNase H1, H2 and the co-precipitated P32 proteins are indicated. The size marker was SeeBlue Plus2 Pre-Stained Standard (Invitrogen). ( C ) 2D gel electrophoresis of proteins co-precipitated with Flag-H1 or Flag-H2. About 5 mg cell lysates were prepared for immunoprecipitation with anti-flag beads from cell lines which stably express Flag-H1 or Flag-H2. The immunoprecipitates were washed four times with RIPA buffer and directly sent to Applied Biomics Inc. (San Francisco, CA) for 2D gel electrophoresis coupled with MS analysis. In brief, the co-precipitated proteins from Flag-H1 or Flag-H2 cells were labeled by fluorescent DIGE CyDyers, respectively, followed by 2D gel electrophoresis. The protein image was scanned with a fluorescence detector. The figure illustrates the proteins differentially associated with RNase H1 (green) or H2 (red). The P32 protein was confirmed with mass spectrum from the extracted gel sample. Circled spots were identified as RNase H1, H2 or P32 by mass spectrometric analysis. ( D ) Both endogenous and expressed RNase H1 are co-precipitated with the expressed P32. Left panel: western blots with P32, RNase H1, or H2 antibodies for proteins co-precipitated using anti-HA antibody from extracts of control HeLa cells or cells transfected with HA-P32 expression plasmid. Right panel: western blots for proteins co-selected using anti-HA antibody from extracts of Flag-H1, Flag-H2 stable cell lines and control cells, all of which were transfected with HA-P32 expression plasmid. ( E ) Confirmation of the specific interaction between RNase H1 and P32. RNase H cleavage activity indicates that the P32 co-immunoprecipitated material contains only RNase H1 enzyme activity. Upper panel: Cleavage patterns of human RNase H1 and H2 from IP-coupled enzyme activity assays. Immunoprecipitations were performed with either anti-flag, anti-RNase H1 or anti-H2 antibodies from extracts of Flag-H1, Flag-H2 expressing cells or control cells. The co-precipitated samples were incubated for the indicated times with a 32 P-labeled RNA/DNA-methoxyethyl (MOE) gapmer duplex and the cleavage products were separated using denaturing gel electrophoresis. The preferred cleavage sites of RNase H1 and H2 are indicated with * or #, respectively. The positions of the preferred cleavage sites in the heteroduplex are shown in the middle panel with the sequences of the RNA substrate (upper strand) and the oligonucleotide (lower strand). The bold nucleotides in the oligonucleotide strand indicate the position of the MOE substitutions. Lower panel: only the RNase H1 enzyme activity was detected in the co-precipitated material from lysates containing tagged P32. Immunoprecipitations were performed with anti-HA antibody from extracts of Flag-H1 or Flag-H2 stable cell lines or control HEK cells, which were all transfected or not transfected with HA-P32 expression plasmid. The precipitated samples were analyzed for cleavage patterns as described above. The position of the cleavage bands relative to the sequence of the cleavage products is shown on the left. A partial alkaline digestion of the same labeled RNA was used as a sequence ladder. The cleavage pattern of purified human RNase H1 is shown at the far right of the lower panel.

    Techniques Used: Western Blot, Immunoprecipitation, Expressing, Stable Transfection, Transformation Assay, Selection, Binding Assay, SDS Page, Silver Staining, Mass Spectrometry, Marker, Staining, Two-Dimensional Gel Electrophoresis, Electrophoresis, Labeling, Fluorescence, Transfection, Plasmid Preparation, Activity Assay, Incubation, Nucleic Acid Electrophoresis, Sequencing, Purification

    15) Product Images from "m5C modification of mRNA serves a DNA damage code to promote homologous recombination"

    Article Title: m5C modification of mRNA serves a DNA damage code to promote homologous recombination

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16722-7

    m 5 C mRNA methylation is enriched at transcriptionally active sites with DNA damage. a U2OS-TRE cells transfected with TA-KR/TA-Cherry/tetR-KR/tetR-Cherry plasmids were exposed to light for 30 min for KR activation and allowed to recover for 1 h before harvest (scale bar: 10 μm). Quantification of frequency of cells in 500 cells with m 5 C foci from three independent experiments, mean ± SD (upper right). Fold increase of m 5 C mean intensity = mean intensity of m 5 C at TA-KR/mean intensity of background ( n = 20, mean ± SD) (lower right). b U2OS-TRE cells were transfected with TA-KR/TA-Cherry to induce local oxidative damage or for the control condition. Cells were then stained for m 5 C with four different anti-m 5 C antibodies. Frequency of m 5 C-positive cells in 500 cells was quantified ( n = 3, mean ± SD). c U2OS-TRE cells transfected with TA-KR were digested with RNaseH1, RNaseA, or DNase I and stained for m 5 C quantification (scale bar: 10 μm). d The mRNA from Flp-in 293 cells treated with or without 2 mM H 2 O 2 for 40 min was used for m 5 C measurement via dot blot. Quantification of m 5 C levels (mean ± SD) from three independent experiments normalized with Ctrl and methylene blue is shown. e 32 P-labeled mRNA monophosphate nucleosides were run on 2D gels for 2D-TLC analysis. In vitro-transcribed 4B mRNA with or without m 5 C was run in parallel. Representative images from three sets of independent experiments are shown with arrows showing the directions of each solvent run. Position of each nucleotide and m 5 C are labeled (Left). f 32 P-labeled mRNA monophosphate nucleosides from U2OS cells with or without 2 mM H 2 O 2 for 40 min were run on 2D gels for 2D-TLC analysis. Representative images from three sets of independent experiments. Associated quantification of relative increase in m 5 C in peroxide-treated cells compared to control, normalized to nucleotide C (right). Statistical analysis was performed with the unpaired two tailed Student’s t -test. * p
    Figure Legend Snippet: m 5 C mRNA methylation is enriched at transcriptionally active sites with DNA damage. a U2OS-TRE cells transfected with TA-KR/TA-Cherry/tetR-KR/tetR-Cherry plasmids were exposed to light for 30 min for KR activation and allowed to recover for 1 h before harvest (scale bar: 10 μm). Quantification of frequency of cells in 500 cells with m 5 C foci from three independent experiments, mean ± SD (upper right). Fold increase of m 5 C mean intensity = mean intensity of m 5 C at TA-KR/mean intensity of background ( n = 20, mean ± SD) (lower right). b U2OS-TRE cells were transfected with TA-KR/TA-Cherry to induce local oxidative damage or for the control condition. Cells were then stained for m 5 C with four different anti-m 5 C antibodies. Frequency of m 5 C-positive cells in 500 cells was quantified ( n = 3, mean ± SD). c U2OS-TRE cells transfected with TA-KR were digested with RNaseH1, RNaseA, or DNase I and stained for m 5 C quantification (scale bar: 10 μm). d The mRNA from Flp-in 293 cells treated with or without 2 mM H 2 O 2 for 40 min was used for m 5 C measurement via dot blot. Quantification of m 5 C levels (mean ± SD) from three independent experiments normalized with Ctrl and methylene blue is shown. e 32 P-labeled mRNA monophosphate nucleosides were run on 2D gels for 2D-TLC analysis. In vitro-transcribed 4B mRNA with or without m 5 C was run in parallel. Representative images from three sets of independent experiments are shown with arrows showing the directions of each solvent run. Position of each nucleotide and m 5 C are labeled (Left). f 32 P-labeled mRNA monophosphate nucleosides from U2OS cells with or without 2 mM H 2 O 2 for 40 min were run on 2D gels for 2D-TLC analysis. Representative images from three sets of independent experiments. Associated quantification of relative increase in m 5 C in peroxide-treated cells compared to control, normalized to nucleotide C (right). Statistical analysis was performed with the unpaired two tailed Student’s t -test. * p

    Techniques Used: Methylation, Transfection, Activation Assay, Staining, Dot Blot, Labeling, Thin Layer Chromatography, In Vitro, Two Tailed Test

    16) Product Images from "Structural rearrangements in the mitochondrial genome of Drosophila melanogaster induced by elevated levels of the replicative DNA helicase"

    Article Title: Structural rearrangements in the mitochondrial genome of Drosophila melanogaster induced by elevated levels of the replicative DNA helicase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky094

    Deletions within the A+T region of mtDNA in Schneider S2 cells overexpressing mtDNA helicase. ( A ) Agarose gel electrophoresis of HindIII-treated mtNA (4 μg) obtained from control (lane 1) and mtDNA helicase-overexpressing (lane 2) S2 cells. The uncut material and resulting HindIII A, B and C fragments are indicated on the right. The bracket shows the distribution of RNase A-sensitive material (see   Supplementary Figure S2 ). Fragment size is indicated in kb on the left. ( B ) Agarose gel electrophoresis of HindIII-SacI-treated mtNA (4 μg) obtained from control (lane 1) and mtDNA helicase-overexpressing (lane 2) S2 cells. The resulting bands are labeled relative to the HindIII fragment nomenclature on the right: uncut material and control HindIII B fragment (lane 1) remain undigested; deletion-bearing HindIII B fragments of mtDNA from helicase-overexpressing cells (lane 2) are labeled as B1, B2, B3; SacI digestion of the HindIII A fragment generates derivative fragments A, A’, A’’; SacI digestion of the HindIII C fragment generates derivative fragments C, C’. Fragment size is indicated in kb on the left. ( C ) Southern-blotting analysis of HindIII-treated mtNA samples obtained from control (lane 1) and mtDNA helicase-overexpressing (lane 2) S2 cells. Fragments encompassing the origin of replication were visualized by hybridization with the Ori probe (see Figure   4A , and Materials and Methods). The HindIII B fragment and its deletion-bearing derivatives B1, B2 and B3, as well as uncut material are labeled on the right. ( D ) Southern-blotting analysis of HindIII-treated mtNA samples obtained from control (lanes 1) and mtDNA helicase-overexpressing (lanes 2) S2 cells. Fragments were visualized by ethidium bromide staining in the agarose gel (EtBr), or by hybridization with probes specific to the replication origin site (Ori), and coding region (6, see Figure   1 ). M indicates a molecular weight standard with fragment sizes indicated in kb at left.
    Figure Legend Snippet: Deletions within the A+T region of mtDNA in Schneider S2 cells overexpressing mtDNA helicase. ( A ) Agarose gel electrophoresis of HindIII-treated mtNA (4 μg) obtained from control (lane 1) and mtDNA helicase-overexpressing (lane 2) S2 cells. The uncut material and resulting HindIII A, B and C fragments are indicated on the right. The bracket shows the distribution of RNase A-sensitive material (see Supplementary Figure S2 ). Fragment size is indicated in kb on the left. ( B ) Agarose gel electrophoresis of HindIII-SacI-treated mtNA (4 μg) obtained from control (lane 1) and mtDNA helicase-overexpressing (lane 2) S2 cells. The resulting bands are labeled relative to the HindIII fragment nomenclature on the right: uncut material and control HindIII B fragment (lane 1) remain undigested; deletion-bearing HindIII B fragments of mtDNA from helicase-overexpressing cells (lane 2) are labeled as B1, B2, B3; SacI digestion of the HindIII A fragment generates derivative fragments A, A’, A’’; SacI digestion of the HindIII C fragment generates derivative fragments C, C’. Fragment size is indicated in kb on the left. ( C ) Southern-blotting analysis of HindIII-treated mtNA samples obtained from control (lane 1) and mtDNA helicase-overexpressing (lane 2) S2 cells. Fragments encompassing the origin of replication were visualized by hybridization with the Ori probe (see Figure 4A , and Materials and Methods). The HindIII B fragment and its deletion-bearing derivatives B1, B2 and B3, as well as uncut material are labeled on the right. ( D ) Southern-blotting analysis of HindIII-treated mtNA samples obtained from control (lanes 1) and mtDNA helicase-overexpressing (lanes 2) S2 cells. Fragments were visualized by ethidium bromide staining in the agarose gel (EtBr), or by hybridization with probes specific to the replication origin site (Ori), and coding region (6, see Figure 1 ). M indicates a molecular weight standard with fragment sizes indicated in kb at left.

    Techniques Used: Agarose Gel Electrophoresis, Labeling, Southern Blot, Hybridization, Staining, Molecular Weight

    17) Product Images from "The RNA binding protein Npl3 promotes resection of DNA double-strand breaks by regulating the levels of Exo1"

    Article Title: The RNA binding protein Npl3 promotes resection of DNA double-strand breaks by regulating the levels of Exo1

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx347

    The lack of NPL3 impairs extensive resection of DSB ends. ( A and B ) Exponentially growing cell cultures of wild type and npl3Δ strains, both carrying a centromeric plasmid either expressing the RNH1 gene from the tetO promoter or empty (vect), were either serially diluted (1:10) before being spotted out onto YEPD plates with or without MMS or CPT (A), or transferred to YEPRG to monitor Rad53 phosphorylation by western blot (B). ( C ) System to detect DSB resection. Gel blots of SspI-digested genomic DNA separated on alkaline agarose gel were hybridized with a single-stranded RNA MAT probe (ss probe) that anneals to the unresected strand. 5΄-3΄ resection progressively eliminates SspI sites (S), producing larger SspI fragments (r1 through r7) detected by the probe. ( D and E ) Exponentially growing YEPR cell cultures were arrested in G2 with nocodazole and transferred to YEPRG (time zero) in the presence of nocodazole. (D) DSB resection as described in (C). (E) Resection products in (D) were analyzed by densitometry. The mean values are represented with error bars denoting SD ( n = 5).
    Figure Legend Snippet: The lack of NPL3 impairs extensive resection of DSB ends. ( A and B ) Exponentially growing cell cultures of wild type and npl3Δ strains, both carrying a centromeric plasmid either expressing the RNH1 gene from the tetO promoter or empty (vect), were either serially diluted (1:10) before being spotted out onto YEPD plates with or without MMS or CPT (A), or transferred to YEPRG to monitor Rad53 phosphorylation by western blot (B). ( C ) System to detect DSB resection. Gel blots of SspI-digested genomic DNA separated on alkaline agarose gel were hybridized with a single-stranded RNA MAT probe (ss probe) that anneals to the unresected strand. 5΄-3΄ resection progressively eliminates SspI sites (S), producing larger SspI fragments (r1 through r7) detected by the probe. ( D and E ) Exponentially growing YEPR cell cultures were arrested in G2 with nocodazole and transferred to YEPRG (time zero) in the presence of nocodazole. (D) DSB resection as described in (C). (E) Resection products in (D) were analyzed by densitometry. The mean values are represented with error bars denoting SD ( n = 5).

    Techniques Used: Plasmid Preparation, Expressing, Cycling Probe Technology, Western Blot, Agarose Gel Electrophoresis

    EXO1 RNA in the absence of Npl3. ( A ) Schematic representation of the EXO1 locus. Primer pairs (PP1-PP6) used for qRT-PCR are indicated by arrows. A bar indicates the 1437 bp-DNA probe internal to the EXO1 coding sequence (+628 to + 2065 from the ATG initiation codon) used for northern blot. ( B ) Total RNA was extracted from exponentially growing YEPD cell cultures of the indicated strains and subjected to quantitative reverse transcriptase PCR (qRT-PCR) with primer pairs located into the EXO1 (PP1 in (A)) and ALG9 coding sequences. The EXO1 RNA levels relative to wild type (set to 1.0) were calculated using ΔΔCt method after normalization to the ALG9 RNA levels for each sample. The mean values are represented with error bars denoting SD ( n = 5). ( C–E ) Total RNA extracted from the indicated cell cultures was subjected to northern blot and hybridized with the probe as in (A). The agarose gels were stained with ethidium bromide to detect 18S and 25S rRNAs (bottom). ( F ) Total RNA extracted from wild type and npl3Δ cells was subjected to 5΄ RACE to visualize the EXO1 5΄ partial cDNA ends. After reverse transcription with a EXO1 specific primer and poly(A) tailing, two subsequent PCR reactions were performed with primers annealing to the appended tail and to the EXO1 coding sequence 718 and 248 bp downstream the EXO1 initiation codon. The final PCR products were separated on a 1.5% agarose gel and visualized with ethidium bromide. ( G ) Total RNA as in (F) was subjected to qRT-PCR with primer pairs depicted in (A), or located in the ALG9 coding sequence. The amount of products obtained with different EXO1 primer pairs was normalized to the ALG9 product using ΔΔCt method. Then, the normalized RNA levels estimated with the different primer pairs in the EXO1 locus were normalized to the RNA levels evaluated with the PP1 primer pair and set to 1.0 for each strain. The mean values are represented with error bars denoting SD ( n = 4).
    Figure Legend Snippet: EXO1 RNA in the absence of Npl3. ( A ) Schematic representation of the EXO1 locus. Primer pairs (PP1-PP6) used for qRT-PCR are indicated by arrows. A bar indicates the 1437 bp-DNA probe internal to the EXO1 coding sequence (+628 to + 2065 from the ATG initiation codon) used for northern blot. ( B ) Total RNA was extracted from exponentially growing YEPD cell cultures of the indicated strains and subjected to quantitative reverse transcriptase PCR (qRT-PCR) with primer pairs located into the EXO1 (PP1 in (A)) and ALG9 coding sequences. The EXO1 RNA levels relative to wild type (set to 1.0) were calculated using ΔΔCt method after normalization to the ALG9 RNA levels for each sample. The mean values are represented with error bars denoting SD ( n = 5). ( C–E ) Total RNA extracted from the indicated cell cultures was subjected to northern blot and hybridized with the probe as in (A). The agarose gels were stained with ethidium bromide to detect 18S and 25S rRNAs (bottom). ( F ) Total RNA extracted from wild type and npl3Δ cells was subjected to 5΄ RACE to visualize the EXO1 5΄ partial cDNA ends. After reverse transcription with a EXO1 specific primer and poly(A) tailing, two subsequent PCR reactions were performed with primers annealing to the appended tail and to the EXO1 coding sequence 718 and 248 bp downstream the EXO1 initiation codon. The final PCR products were separated on a 1.5% agarose gel and visualized with ethidium bromide. ( G ) Total RNA as in (F) was subjected to qRT-PCR with primer pairs depicted in (A), or located in the ALG9 coding sequence. The amount of products obtained with different EXO1 primer pairs was normalized to the ALG9 product using ΔΔCt method. Then, the normalized RNA levels estimated with the different primer pairs in the EXO1 locus were normalized to the RNA levels evaluated with the PP1 primer pair and set to 1.0 for each strain. The mean values are represented with error bars denoting SD ( n = 4).

    Techniques Used: Quantitative RT-PCR, Sequencing, Northern Blot, Polymerase Chain Reaction, Staining, Agarose Gel Electrophoresis

    18) Product Images from "The Hepatitis B Virus Ribonuclease H Is Sensitive to Inhibitors of the Human Immunodeficiency Virus Ribonuclease H and Integrase Enzymes"

    Article Title: The Hepatitis B Virus Ribonuclease H Is Sensitive to Inhibitors of the Human Immunodeficiency Virus Ribonuclease H and Integrase Enzymes

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003125

    Specificity of anti-HBV RNAseH compounds. A. Inhibition of HBV genotype D RNAseH by irrelevant compounds at 10 µM in the oligonucleotide-directed RNAseH assay. Compound #4 was employed as an example HBV RNAseH inhibitor. B. Anti-HBV RNAseH inhibitors do not significantly inhibit the HCV RNA polymerase. The ability of compounds #5, 6 and 8 to inhibit production of poly-G by the HCV RNA-directed RNA polymerase was measured in a primed homopolymeric RNA synthesis assay [82] . The compounds were employed at 10 µM. DMSO, vehicle control. C. Dose-responsiveness of HBV RNAseH inhibition. The effects of compounds #6, 8, and 12 on the RNAseH activity of HRHPL (genotype D) were measured at concentrations ranging from 0.5 to 50 µM. The dose-response profile is plotted for compound #12.
    Figure Legend Snippet: Specificity of anti-HBV RNAseH compounds. A. Inhibition of HBV genotype D RNAseH by irrelevant compounds at 10 µM in the oligonucleotide-directed RNAseH assay. Compound #4 was employed as an example HBV RNAseH inhibitor. B. Anti-HBV RNAseH inhibitors do not significantly inhibit the HCV RNA polymerase. The ability of compounds #5, 6 and 8 to inhibit production of poly-G by the HCV RNA-directed RNA polymerase was measured in a primed homopolymeric RNA synthesis assay [82] . The compounds were employed at 10 µM. DMSO, vehicle control. C. Dose-responsiveness of HBV RNAseH inhibition. The effects of compounds #6, 8, and 12 on the RNAseH activity of HRHPL (genotype D) were measured at concentrations ranging from 0.5 to 50 µM. The dose-response profile is plotted for compound #12.

    Techniques Used: Inhibition, Activity Assay

    Recombinant RNAseHs from HBV genotypes A, B, C, D, and H. A. Sequence alignment for genotype A, B, C, D, and H versions of the HBV RNAseH expression construct HRHPL. The additional methionine at residue 10 of the genotype D sequence is a product of the cloning strategy; this insertion has no impact on the RNAseH activity because the first 9 amino acids of HRHPL can be deleted without altering the biochemical profile of the enzyme. * indicates the DEDD active site residues, and the hexahistidine tag at the C-terminus is underlined. Residue 1 for the HBV RNAseH domain is amino acid 684 in the full-length polymerase protein (strain adw2). B. Western analysis of RNAseH proteins in the enriched lysates probed with the anti-HBV RNAseH monoclonal antibody 9F9. C. RNAseH activity of RNAseH from HBV genotypes A, B, C, D, and H detected by the oligonucleotide-directed RNA cleavage assay. HRHPL-D702A (genotype D) is a negative control. gt, genotype.
    Figure Legend Snippet: Recombinant RNAseHs from HBV genotypes A, B, C, D, and H. A. Sequence alignment for genotype A, B, C, D, and H versions of the HBV RNAseH expression construct HRHPL. The additional methionine at residue 10 of the genotype D sequence is a product of the cloning strategy; this insertion has no impact on the RNAseH activity because the first 9 amino acids of HRHPL can be deleted without altering the biochemical profile of the enzyme. * indicates the DEDD active site residues, and the hexahistidine tag at the C-terminus is underlined. Residue 1 for the HBV RNAseH domain is amino acid 684 in the full-length polymerase protein (strain adw2). B. Western analysis of RNAseH proteins in the enriched lysates probed with the anti-HBV RNAseH monoclonal antibody 9F9. C. RNAseH activity of RNAseH from HBV genotypes A, B, C, D, and H detected by the oligonucleotide-directed RNA cleavage assay. HRHPL-D702A (genotype D) is a negative control. gt, genotype.

    Techniques Used: Recombinant, Sequencing, Expressing, Construct, Clone Assay, Activity Assay, Western Blot, Cleavage Assay, Negative Control

    Recombinant HBV RNAseH is enzymatically active. A. Oligonucleotide-directed RNAseH assay. Uniformly 32 P-labeled RNA (blue or red) is annealed to a complementary DNA oligonucleotide (black). RNAseH activity cleaves the RNA in the heteroduplex formed where the oligonucleotide anneals to the RNA and yields two products (P1 and P2). B. Recombinant HBV RNAseH is active. An oligonucleotide-directed RNAseH assay was conducted with E. coli RNAseH, wild-type HBV RNAseH (HRHPL), or RNAseH-deficient HRHPL (D702A). A complementary oligonucleotide (+) or non-complementary oligonucleotide (−) was mixed with labeled DRF+ RNA and the reactions were incubated to allow RNAseH activity. The products were resolved by SDS-PAGE and the RNAs were detected by autoradiography. Oligonucleotide set 1 was D2507− and D2526+ and oligonucleotide set #2 was D2543M-Sal and D2453+. The positions of the cleavage products (P1 and P2) are indicated in blue for reactions containing oligonucleotide D2507− and in red for reactions containing oligonucleotide D2543M-Sal. C. FRET-based RNAseH assay. A self-complementary chimeric RNA:DNA synthetic oligonucleotide (RHF1) forms a stem-loop in which the stem is an RNA:DNA heteroduplex. The stem brings the fluorescein (F) and quencher (Q) at the 5′ and 3′ ends of the oligonucleotide into close proximity. Cleavage of the RNA releases the fluorescein and increases its fluorescence. D. Detection of HBV RNAseH activity employing the fluorescent assay. The substrate in panel C was employed in an RNAseH assay employing buffer alone, wild-type HBV RNAseH (HRHPL), or RNAseH-deficient HRHPL (D702A/E731A). *, P
    Figure Legend Snippet: Recombinant HBV RNAseH is enzymatically active. A. Oligonucleotide-directed RNAseH assay. Uniformly 32 P-labeled RNA (blue or red) is annealed to a complementary DNA oligonucleotide (black). RNAseH activity cleaves the RNA in the heteroduplex formed where the oligonucleotide anneals to the RNA and yields two products (P1 and P2). B. Recombinant HBV RNAseH is active. An oligonucleotide-directed RNAseH assay was conducted with E. coli RNAseH, wild-type HBV RNAseH (HRHPL), or RNAseH-deficient HRHPL (D702A). A complementary oligonucleotide (+) or non-complementary oligonucleotide (−) was mixed with labeled DRF+ RNA and the reactions were incubated to allow RNAseH activity. The products were resolved by SDS-PAGE and the RNAs were detected by autoradiography. Oligonucleotide set 1 was D2507− and D2526+ and oligonucleotide set #2 was D2543M-Sal and D2453+. The positions of the cleavage products (P1 and P2) are indicated in blue for reactions containing oligonucleotide D2507− and in red for reactions containing oligonucleotide D2543M-Sal. C. FRET-based RNAseH assay. A self-complementary chimeric RNA:DNA synthetic oligonucleotide (RHF1) forms a stem-loop in which the stem is an RNA:DNA heteroduplex. The stem brings the fluorescein (F) and quencher (Q) at the 5′ and 3′ ends of the oligonucleotide into close proximity. Cleavage of the RNA releases the fluorescein and increases its fluorescence. D. Detection of HBV RNAseH activity employing the fluorescent assay. The substrate in panel C was employed in an RNAseH assay employing buffer alone, wild-type HBV RNAseH (HRHPL), or RNAseH-deficient HRHPL (D702A/E731A). *, P

    Techniques Used: Recombinant, Labeling, Activity Assay, Incubation, SDS Page, Autoradiography, Fluorescence

    19) Product Images from "RNase H1 Regulates Mitochondrial Transcription and Translation via the Degradation of 7S RNA"

    Article Title: RNase H1 Regulates Mitochondrial Transcription and Translation via the Degradation of 7S RNA

    Journal: Frontiers in Genetics

    doi: 10.3389/fgene.2019.01393

    RNASEH1 mutations, transcript, and protein levels. (A) Domains ofhuman RNAse H1 protein (MTS, mitochondrial targeting sequence; HBD, hybrid binding domain; CD, connection domain; catalytic domain). RNase H1 protein sequences from representative species, H. sapiens (Hs, NP_002927) M. musculus (Mm, NP_035405), B. taurus (Bt, NP_001039970), G. gallus (Gg, NP_990329), X. tropicalis (Xt, NP_001096299), D. rerio (Dr, NP_001002659), C. intestinalis (Ci, F6QPH0), D. melanogaster (Dm, NP_995777), C. elegans (Ce, NP_001040786), S. cerevisiae (Sc, Q04740), were extracted from the database and aligned using ClustalW2. Conserved residues found mutated in the patient in exon 4 are boxed in red, while residues in the active site and interacting with DNA or RNA are boxed in yellow and green, respectively. Positions of β strands are marked by blue arrows. (B) Human RNase H1 crystal structure (PDB ID 2QK9) 18 bp DNA(cyan): RNA (magenta) hybrid is shown respectively. Residues in the active site are colored in yellow. Residues Trp 164 (green) and Val 142 , or the mutated variant Ile 142 (red), are shown as sticks. (C) RNASEH1 transcript levels in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium assessed by qPCR (probe Hs00268000_m1) and normalized to GAPDH transcript levels. Data are shown as mean ± SD, n = 4, ***p
    Figure Legend Snippet: RNASEH1 mutations, transcript, and protein levels. (A) Domains ofhuman RNAse H1 protein (MTS, mitochondrial targeting sequence; HBD, hybrid binding domain; CD, connection domain; catalytic domain). RNase H1 protein sequences from representative species, H. sapiens (Hs, NP_002927) M. musculus (Mm, NP_035405), B. taurus (Bt, NP_001039970), G. gallus (Gg, NP_990329), X. tropicalis (Xt, NP_001096299), D. rerio (Dr, NP_001002659), C. intestinalis (Ci, F6QPH0), D. melanogaster (Dm, NP_995777), C. elegans (Ce, NP_001040786), S. cerevisiae (Sc, Q04740), were extracted from the database and aligned using ClustalW2. Conserved residues found mutated in the patient in exon 4 are boxed in red, while residues in the active site and interacting with DNA or RNA are boxed in yellow and green, respectively. Positions of β strands are marked by blue arrows. (B) Human RNase H1 crystal structure (PDB ID 2QK9) 18 bp DNA(cyan): RNA (magenta) hybrid is shown respectively. Residues in the active site are colored in yellow. Residues Trp 164 (green) and Val 142 , or the mutated variant Ile 142 (red), are shown as sticks. (C) RNASEH1 transcript levels in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium assessed by qPCR (probe Hs00268000_m1) and normalized to GAPDH transcript levels. Data are shown as mean ± SD, n = 4, ***p

    Techniques Used: Sequencing, Binding Assay, Variant Assay, Real-time Polymerase Chain Reaction

    20) Product Images from "Antisense oligonucleotides targeting translation inhibitory elements in 5′ UTRs can selectively increase protein levels"

    Article Title: Antisense oligonucleotides targeting translation inhibitory elements in 5′ UTRs can selectively increase protein levels

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx632

    ASOs targeting the 5′ UTR of RNASEH1 mRNA increase protein production. ( A ) Predicted secondary structure of the 5′ UTR of RNASEH1 . The upper case letters indicate coding sequence. The start codon of the uORF is highlighted in blue. Binding sites for ASOs are indicated by lines. ( B ) Western blot for RNASEH1 in HeLa cells treated with indicated ASOs for 15 h at 25 nM. Numbers below the lanes are percentages of RNASEH1 protein relative to mock-treated cells; values are normalized to quantity of tubulin loading. ( C ) Schematic representation of ASO positions on RNASEH1 bracketing active ASO761919. ( D ) Western blot for RNASEH1 in HeLa cells treated for 15 h with 25 nM indicated ASOs. GAPDH was used as a loading control. ( E ) Sequences of RNASEH1 mRNA and ASOs with mismatches (underlined). ( F ) Western analysis for RNASEH1 protein in HeLa cells treated with 30 nM indicated ASOs for 10 h. P32 was detected as a loading control. ( G ) Western analyses for RNASEH1 in HeLa cells co-transfected with the RNASEH1 ASO761919 and an ASO complementary to the RNASEH1 ASO (ASO927728). ASO concentration 0 indicates mock transfection. ( H ) Western blot analyses for RNASEH1 in HeLa cells co-transfected for 10 h with ASO761919 and a control ASO759704. The mean values and standard deviations after normalization to P32 quantity are shown below the lanes. ( I ) qRT-PCR quantification of RNASEH1 mRNA in cells treated with indicated ASOs. The error bars represent standard deviations from three experiments. P -values were calculated based on unpaired t-test. NS, not significant. ** P
    Figure Legend Snippet: ASOs targeting the 5′ UTR of RNASEH1 mRNA increase protein production. ( A ) Predicted secondary structure of the 5′ UTR of RNASEH1 . The upper case letters indicate coding sequence. The start codon of the uORF is highlighted in blue. Binding sites for ASOs are indicated by lines. ( B ) Western blot for RNASEH1 in HeLa cells treated with indicated ASOs for 15 h at 25 nM. Numbers below the lanes are percentages of RNASEH1 protein relative to mock-treated cells; values are normalized to quantity of tubulin loading. ( C ) Schematic representation of ASO positions on RNASEH1 bracketing active ASO761919. ( D ) Western blot for RNASEH1 in HeLa cells treated for 15 h with 25 nM indicated ASOs. GAPDH was used as a loading control. ( E ) Sequences of RNASEH1 mRNA and ASOs with mismatches (underlined). ( F ) Western analysis for RNASEH1 protein in HeLa cells treated with 30 nM indicated ASOs for 10 h. P32 was detected as a loading control. ( G ) Western analyses for RNASEH1 in HeLa cells co-transfected with the RNASEH1 ASO761919 and an ASO complementary to the RNASEH1 ASO (ASO927728). ASO concentration 0 indicates mock transfection. ( H ) Western blot analyses for RNASEH1 in HeLa cells co-transfected for 10 h with ASO761919 and a control ASO759704. The mean values and standard deviations after normalization to P32 quantity are shown below the lanes. ( I ) qRT-PCR quantification of RNASEH1 mRNA in cells treated with indicated ASOs. The error bars represent standard deviations from three experiments. P -values were calculated based on unpaired t-test. NS, not significant. ** P

    Techniques Used: Sequencing, Binding Assay, Western Blot, Allele-specific Oligonucleotide, Transfection, Concentration Assay, Quantitative RT-PCR

    The ACP1 ASO enhances binding of translation initiation factors to ACP1 mRNA. ( A ) Western analyses of ACP1 and LDLR proteins in HeLa cells treated for 30 h with 15 μM 4E1Rcat. The lower panel shows a Coomassie-blue stained image from a duplicate gel, which serves as control to ensure equal loading. ( B ) Western analysis for DHX29 in HEK293 cells treated with 3 nM siRNAs targeting DHX29 or luc for 24 h. ( C ) Western analysis for ACP1 protein in HEK293 cells treated with DHX29 or luc siRNAs for 24 h, followed by transfection of ASO812653 for an additional 10 h. ANXA2 served as a loading control. The percentages of ACP1 protein relative to mock treated cells (ASO concentration 0) are shown below the lanes. ( D ) Western analyses for eIF4A (upper panel) and eIF2a (middle panel) proteins co-isolated with a 3′-biotinylated, 5′-capped RNA derived from the 5′ UTR of human ACP1 mRNA in the presence of ASO812658, control ASO XL398, or no ASO. The same membrane was re-probed using rabbit serum to detect non-specific band that served as loading control (lower panel, indicated by an asterisk). Percentages relative to samples with no ASO are given. ( E ) qRT-PCR quantification of ACP1 mRNA (left panel), 7SL RNA (middle panel), and ACTB mRNA (right panel) co-immunoprecipitated using an anti-eIF4A antibody or a control IgG from cells transfected with indicated ASOs or mock-transfected cells (UTC). Plotted are means ± standard deviations from three experiments. P -values were calculated based on unpaired t -test. NS, not significant. * P
    Figure Legend Snippet: The ACP1 ASO enhances binding of translation initiation factors to ACP1 mRNA. ( A ) Western analyses of ACP1 and LDLR proteins in HeLa cells treated for 30 h with 15 μM 4E1Rcat. The lower panel shows a Coomassie-blue stained image from a duplicate gel, which serves as control to ensure equal loading. ( B ) Western analysis for DHX29 in HEK293 cells treated with 3 nM siRNAs targeting DHX29 or luc for 24 h. ( C ) Western analysis for ACP1 protein in HEK293 cells treated with DHX29 or luc siRNAs for 24 h, followed by transfection of ASO812653 for an additional 10 h. ANXA2 served as a loading control. The percentages of ACP1 protein relative to mock treated cells (ASO concentration 0) are shown below the lanes. ( D ) Western analyses for eIF4A (upper panel) and eIF2a (middle panel) proteins co-isolated with a 3′-biotinylated, 5′-capped RNA derived from the 5′ UTR of human ACP1 mRNA in the presence of ASO812658, control ASO XL398, or no ASO. The same membrane was re-probed using rabbit serum to detect non-specific band that served as loading control (lower panel, indicated by an asterisk). Percentages relative to samples with no ASO are given. ( E ) qRT-PCR quantification of ACP1 mRNA (left panel), 7SL RNA (middle panel), and ACTB mRNA (right panel) co-immunoprecipitated using an anti-eIF4A antibody or a control IgG from cells transfected with indicated ASOs or mock-transfected cells (UTC). Plotted are means ± standard deviations from three experiments. P -values were calculated based on unpaired t -test. NS, not significant. * P

    Techniques Used: Allele-specific Oligonucleotide, Binding Assay, Western Blot, Staining, Transfection, Concentration Assay, Isolation, Derivative Assay, Quantitative RT-PCR, Immunoprecipitation

    21) Product Images from "Dynamic nucleoplasmic and nucleolar localization of mammalian RNase H1 in response to RNAP I transcriptional R-loops"

    Article Title: Dynamic nucleoplasmic and nucleolar localization of mammalian RNase H1 in response to RNAP I transcriptional R-loops

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx710

    Top1 levels are increased in Rnaseh1-knockout mice to reduce nucleolar accumulation of R-loops. ( A ) qRT-PCR of Cdkn1a/p21 and Tnfrsf10b from isolated primary mouse hepatocytes. 10 animals per group were assayed. ( B ) Western analysis of RNase H1, p21 and Top1 in liver lysates from control mice and Rnaseh1 -knockout mice that were either 6–7 weeks old (two animals/group) or 9–10 weeks old (three animals/group). ( C ) Representative images of immunofluorescent staining of S9.6 in isolated mouse hepatocytes.
    Figure Legend Snippet: Top1 levels are increased in Rnaseh1-knockout mice to reduce nucleolar accumulation of R-loops. ( A ) qRT-PCR of Cdkn1a/p21 and Tnfrsf10b from isolated primary mouse hepatocytes. 10 animals per group were assayed. ( B ) Western analysis of RNase H1, p21 and Top1 in liver lysates from control mice and Rnaseh1 -knockout mice that were either 6–7 weeks old (two animals/group) or 9–10 weeks old (three animals/group). ( C ) Representative images of immunofluorescent staining of S9.6 in isolated mouse hepatocytes.

    Techniques Used: Knock-Out, Mouse Assay, Quantitative RT-PCR, Isolation, Western Blot, Staining

    22) Product Images from "Human RNase H1 Is Associated with Protein P32 and Is Involved in Mitochondrial Pre-rRNA Processing"

    Article Title: Human RNase H1 Is Associated with Protein P32 and Is Involved in Mitochondrial Pre-rRNA Processing

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0071006

    Both RNase H1 and P32 interact with mitochondrial DNA and pre-rRNA. ( A ) The positions of Probes and PCR primers for the human mitochondrial DNA. The DNA map was derived from published review [65] . Two oligonucleotide probes specific to 12 S and 16 S mitochondria rRNA regions are shown in Blue bars . Three sets of PCR probes corresponding to the A, B and C regions are indicated in Green arrows . ( B ) RNase H1 and P32 bind mitochondrial DNA. Cell extracts were prepared from an HA-H1 stably expressing cell line (RNase H1), control HEK cells or HEK cells transfected with the HA-P32 expression plasmid (P32). Equal amounts of each extract were used for immunoprecipitation with anti-HA beads. Nucleic acids were extracted from the precipitated samples using phenol/chloroform and subjected to PCR analysis. The probe sets for PCR were shown in Figure 6A . Genomic DNA from HEK cells that was used as a positive control. The PCR products were analyzed on 2% Agarose gels. ( C ) RNase H1 may interact with the mitochondrial rDNA region. The extracts from HA-H1 cell and control HEK cells were used for immunoprecipitation with HA-antibody. The precipitates were digested on beads with (+) or without (−) DNase I. The DNA associated with beads was then extracted and subjected to PCR analysis. The PCR products were separated in 2% agarose gel. ( D ) RNase H1 and P32 also co-immunoprecipitated with mitochondrial pre-rRNA. The same extracted nucleic acids from panel B were digested with DNase I. The RNA is used for reverse transcription with (+) or without (−) reverse transcriptase, followed by PCR amplification using different primer sets as indicated below the panels. PCR reaction using primers specific to U16 snoRNA was used as control.
    Figure Legend Snippet: Both RNase H1 and P32 interact with mitochondrial DNA and pre-rRNA. ( A ) The positions of Probes and PCR primers for the human mitochondrial DNA. The DNA map was derived from published review [65] . Two oligonucleotide probes specific to 12 S and 16 S mitochondria rRNA regions are shown in Blue bars . Three sets of PCR probes corresponding to the A, B and C regions are indicated in Green arrows . ( B ) RNase H1 and P32 bind mitochondrial DNA. Cell extracts were prepared from an HA-H1 stably expressing cell line (RNase H1), control HEK cells or HEK cells transfected with the HA-P32 expression plasmid (P32). Equal amounts of each extract were used for immunoprecipitation with anti-HA beads. Nucleic acids were extracted from the precipitated samples using phenol/chloroform and subjected to PCR analysis. The probe sets for PCR were shown in Figure 6A . Genomic DNA from HEK cells that was used as a positive control. The PCR products were analyzed on 2% Agarose gels. ( C ) RNase H1 may interact with the mitochondrial rDNA region. The extracts from HA-H1 cell and control HEK cells were used for immunoprecipitation with HA-antibody. The precipitates were digested on beads with (+) or without (−) DNase I. The DNA associated with beads was then extracted and subjected to PCR analysis. The PCR products were separated in 2% agarose gel. ( D ) RNase H1 and P32 also co-immunoprecipitated with mitochondrial pre-rRNA. The same extracted nucleic acids from panel B were digested with DNase I. The RNA is used for reverse transcription with (+) or without (−) reverse transcriptase, followed by PCR amplification using different primer sets as indicated below the panels. PCR reaction using primers specific to U16 snoRNA was used as control.

    Techniques Used: Polymerase Chain Reaction, Derivative Assay, Stable Transfection, Expressing, Transfection, Plasmid Preparation, Immunoprecipitation, Positive Control, Agarose Gel Electrophoresis, Amplification

    Depletion of RNase H1 or P32 resulted in accumulation of mitochondrial pre-12S/16S rRNA. HeLa cells were treated with 2 nM or 20 nM of RNase H1-siRNA or P32 –siRNA for 24 or 48 hours. ( A ) The mRNA levels of RNase H1 and P32 were determined by qRT-PCR 24 hrs after siRNA treatment. ( B ) Protein levels of RNase H1 and P32 were analyzed by western analysis 24 hours post siRNA treatment. ( C ) Reduction of RNase H1 or P32 significantly increased the level of mitochondrial pre-rRNA. HeLa cells were treated with either RNase H1-siRNA (2 nM) or P32-siRNA (2 nM) for 24 hours. Total RNA was prepared and subjected to Northern analysis with 32 P labeled probes specific to 12S or 16S rRNAs. U3 snoRNA was detected and served as a control. The relative levels of pre-rRNA were measured from the results obtained with 12 S probe, normalized to U3, and plotted in the right panel. The error bars indicate standard error of the three replicates. (D) RT-PCR assay for the levels of pre-16 S and pre-ND3 RNAs. Total RNA prepared from HeLa cells treated for 24 hrs with corresponding siRNAs was analyzed by qRT-PCR, using primer probe sets specific to the tRNA Val -16 S rRNA junction (pre-16 S) or to the tRNA Gly -ND3 junction (pre-ND3). The error bars represent standard deviation of three replicates.
    Figure Legend Snippet: Depletion of RNase H1 or P32 resulted in accumulation of mitochondrial pre-12S/16S rRNA. HeLa cells were treated with 2 nM or 20 nM of RNase H1-siRNA or P32 –siRNA for 24 or 48 hours. ( A ) The mRNA levels of RNase H1 and P32 were determined by qRT-PCR 24 hrs after siRNA treatment. ( B ) Protein levels of RNase H1 and P32 were analyzed by western analysis 24 hours post siRNA treatment. ( C ) Reduction of RNase H1 or P32 significantly increased the level of mitochondrial pre-rRNA. HeLa cells were treated with either RNase H1-siRNA (2 nM) or P32-siRNA (2 nM) for 24 hours. Total RNA was prepared and subjected to Northern analysis with 32 P labeled probes specific to 12S or 16S rRNAs. U3 snoRNA was detected and served as a control. The relative levels of pre-rRNA were measured from the results obtained with 12 S probe, normalized to U3, and plotted in the right panel. The error bars indicate standard error of the three replicates. (D) RT-PCR assay for the levels of pre-16 S and pre-ND3 RNAs. Total RNA prepared from HeLa cells treated for 24 hrs with corresponding siRNAs was analyzed by qRT-PCR, using primer probe sets specific to the tRNA Val -16 S rRNA junction (pre-16 S) or to the tRNA Gly -ND3 junction (pre-ND3). The error bars represent standard deviation of three replicates.

    Techniques Used: Quantitative RT-PCR, Western Blot, Northern Blot, Labeling, Reverse Transcription Polymerase Chain Reaction, Standard Deviation

    Co-localization of P32 and RNase H1. ( A ) Immunofluorescence Staining of P32 and RNase H1. Upper panel: HeLa cells were stained for endogenous P32 and RNase H1 using mouse monoclonal anti-P32 antibody and rabbit anti-RNase H1 antibody, respectively, followed by FITC conjugated donkey anti-mouse ( green ) and TRITC conjugated anti-rabbit secondary antibodies ( red ). Nuclei were stained with DAP1 ( Blue ) and Mitochondria were stained with mitotracker ( white ). Lower panel: HeLa cells were infected with adenovirus expressing RNase H1. Cells were stained as described in upper panel. ( B ) Subcellular fractionation of P32 protein. The proteins from sub-cellular compartments (cytosol, mitochondrial and ER membranes, nucleus and cytoskeleton) were prepared from HEK cells using proteome cell compartment kit (Qiagen). About 10 µg protein samples from each fraction were analyzed by western for P32. The same blot was stripped and tubulin-γ was detected to serve as a control.
    Figure Legend Snippet: Co-localization of P32 and RNase H1. ( A ) Immunofluorescence Staining of P32 and RNase H1. Upper panel: HeLa cells were stained for endogenous P32 and RNase H1 using mouse monoclonal anti-P32 antibody and rabbit anti-RNase H1 antibody, respectively, followed by FITC conjugated donkey anti-mouse ( green ) and TRITC conjugated anti-rabbit secondary antibodies ( red ). Nuclei were stained with DAP1 ( Blue ) and Mitochondria were stained with mitotracker ( white ). Lower panel: HeLa cells were infected with adenovirus expressing RNase H1. Cells were stained as described in upper panel. ( B ) Subcellular fractionation of P32 protein. The proteins from sub-cellular compartments (cytosol, mitochondrial and ER membranes, nucleus and cytoskeleton) were prepared from HEK cells using proteome cell compartment kit (Qiagen). About 10 µg protein samples from each fraction were analyzed by western for P32. The same blot was stripped and tubulin-γ was detected to serve as a control.

    Techniques Used: Immunofluorescence, Staining, Infection, Expressing, Fractionation, Western Blot

    Recombinant P32 binds to recombinant RNase H1, enhances its turnover rate, and reduces the binding affinity of the enzyme for the heteroduplex substrate. ( A ) Coomassie blue staining of the purified human His-H1, GST protein, and GST-P32 proteins separated by SDS-PAGE. The sizes for the standard protein markers are indicated. ( B ) RNase H1 but not P32 appears to bind the heteroduplex substrate. Gel shift assay was performed using 0.4 ug purified RNase H1, GST-P32, or GST proteins incubated at 4°C for 30 min with a non-cleavable heteroduplex containing 32 P labeled uniformly modified 2′-fluoro RNA annealed to DNA and subjected to native gel electrophoresis. ( C ) The interaction between RNase H1 and P32 appears to be equal molar. A fixed amount of GST-P32 was bound to GST affinity beads and then incubated with increasing amounts of RNase H1. Glutathione (GSH) eluted RNase H1 and P32 were quantified by Western blot as described in the Material and Methods. The amounts of bead-bound P32 and P32-associated RNase H1 were determined by loading known amounts of the respective proteins (left panel). The molecular ratio of bound RNase H1 relative to P32 was calculated and plotted in the right panel. ( D ) The effects of ionic strength on RNase H1/P32 interaction. Left panel: RNase H1 binds GST-P32 but not GST protein. GST or GST-P32 bound to anti-GST beads was incubated with RNase H1 in NaCl concentrations ranging from 0-950 mM as described in the Material and Methods. Middle panel: increasing NaCl concentration inhibits binding of RNase H1 to P32. Both unbound (flow through) and bound (affinity eluted) fractions were collected and the levels of RNase H1 and P32 evaluated by western blot. Right panel: Increasing pH reduced binding of RNase H1 to P32. ( E ) Michaelis-Menten kinetics and binding constants for RNase H1 cleavage of an RNA/DNA duplex in the presence or absence of P32. The K m , V max , and K d were determined by incubating the Apo B RNA/DNA duplex with RNase H1 plus GST (as control) or RNase H1 plus different amounts of P32 resulting in an H1:P32 ratio = 1∶1 or 1∶5. An uncleavable competitive inhibitor (2′-fluororibonucleotide/DNA) was used to determine the binding to the RNA/DNA duplex, as described in the Material and Methods. The calculated constants are indicated in the right panel. The error bars indicate the standard error from three parallel experiments.
    Figure Legend Snippet: Recombinant P32 binds to recombinant RNase H1, enhances its turnover rate, and reduces the binding affinity of the enzyme for the heteroduplex substrate. ( A ) Coomassie blue staining of the purified human His-H1, GST protein, and GST-P32 proteins separated by SDS-PAGE. The sizes for the standard protein markers are indicated. ( B ) RNase H1 but not P32 appears to bind the heteroduplex substrate. Gel shift assay was performed using 0.4 ug purified RNase H1, GST-P32, or GST proteins incubated at 4°C for 30 min with a non-cleavable heteroduplex containing 32 P labeled uniformly modified 2′-fluoro RNA annealed to DNA and subjected to native gel electrophoresis. ( C ) The interaction between RNase H1 and P32 appears to be equal molar. A fixed amount of GST-P32 was bound to GST affinity beads and then incubated with increasing amounts of RNase H1. Glutathione (GSH) eluted RNase H1 and P32 were quantified by Western blot as described in the Material and Methods. The amounts of bead-bound P32 and P32-associated RNase H1 were determined by loading known amounts of the respective proteins (left panel). The molecular ratio of bound RNase H1 relative to P32 was calculated and plotted in the right panel. ( D ) The effects of ionic strength on RNase H1/P32 interaction. Left panel: RNase H1 binds GST-P32 but not GST protein. GST or GST-P32 bound to anti-GST beads was incubated with RNase H1 in NaCl concentrations ranging from 0-950 mM as described in the Material and Methods. Middle panel: increasing NaCl concentration inhibits binding of RNase H1 to P32. Both unbound (flow through) and bound (affinity eluted) fractions were collected and the levels of RNase H1 and P32 evaluated by western blot. Right panel: Increasing pH reduced binding of RNase H1 to P32. ( E ) Michaelis-Menten kinetics and binding constants for RNase H1 cleavage of an RNA/DNA duplex in the presence or absence of P32. The K m , V max , and K d were determined by incubating the Apo B RNA/DNA duplex with RNase H1 plus GST (as control) or RNase H1 plus different amounts of P32 resulting in an H1:P32 ratio = 1∶1 or 1∶5. An uncleavable competitive inhibitor (2′-fluororibonucleotide/DNA) was used to determine the binding to the RNA/DNA duplex, as described in the Material and Methods. The calculated constants are indicated in the right panel. The error bars indicate the standard error from three parallel experiments.

    Techniques Used: Recombinant, Binding Assay, Staining, Purification, SDS Page, Electrophoretic Mobility Shift Assay, Incubation, Labeling, Modification, Nucleic Acid Electrophoresis, Western Blot, Concentration Assay, Flow Cytometry

    P32 appears to interact with the N-terminal duplex binding domain of RNase H1. ( A ) Expression and purification of RNase H1 deletion mutants. Left panel: Schematic depiction of the different human RNase H1 deletion mutants. DL1 deletes the hybrid binding domain (amino acid positions 1–73); DL2 deletes both the hybrid binding domain and the spacer domain (amino acid 1–129). The black bars at the N-terminus of each mutant represent a His tag. Right panel: Coomassie blue staining of the purified RNase H1 deletion mutants. The sizes of the standard markers are given. ( B ) Interaction of full length RNase H1 and its deletion mutants with P32. The full length or truncated RNase H1 proteins were incubated with GST-P32 bound to GST-beads under different NaCl concentrations ranging from 150–450 mM in both the binding and washing solutions. The P32 and RNase H1 or deletion mutants were eluted and analyzed by Western blot, using P32 or RNase H1 antibodies, respectively (right panel). Western blot to RNase H1 and deletion mutants DL1 and DL2 demonstrates that the mutant proteins are recognized by the RNase H1 antibody (left panel). ( C ) Michaelis-Menten Kinetics of DL-1 mutant in the presence or absence of P32. K m , V max , and k cat for DL-1 plus GST or GST-P32 (DL-1:P32 = 1:5 in molecular ratio) were determined in 50 and 150 mM NaCl concentration with the Apo B RNA/DNA duplex as described in the Material and Methods.
    Figure Legend Snippet: P32 appears to interact with the N-terminal duplex binding domain of RNase H1. ( A ) Expression and purification of RNase H1 deletion mutants. Left panel: Schematic depiction of the different human RNase H1 deletion mutants. DL1 deletes the hybrid binding domain (amino acid positions 1–73); DL2 deletes both the hybrid binding domain and the spacer domain (amino acid 1–129). The black bars at the N-terminus of each mutant represent a His tag. Right panel: Coomassie blue staining of the purified RNase H1 deletion mutants. The sizes of the standard markers are given. ( B ) Interaction of full length RNase H1 and its deletion mutants with P32. The full length or truncated RNase H1 proteins were incubated with GST-P32 bound to GST-beads under different NaCl concentrations ranging from 150–450 mM in both the binding and washing solutions. The P32 and RNase H1 or deletion mutants were eluted and analyzed by Western blot, using P32 or RNase H1 antibodies, respectively (right panel). Western blot to RNase H1 and deletion mutants DL1 and DL2 demonstrates that the mutant proteins are recognized by the RNase H1 antibody (left panel). ( C ) Michaelis-Menten Kinetics of DL-1 mutant in the presence or absence of P32. K m , V max , and k cat for DL-1 plus GST or GST-P32 (DL-1:P32 = 1:5 in molecular ratio) were determined in 50 and 150 mM NaCl concentration with the Apo B RNA/DNA duplex as described in the Material and Methods.

    Techniques Used: Binding Assay, Expressing, Purification, Mutagenesis, Staining, Incubation, Western Blot, Concentration Assay

    Human RNase H1 is associated with P32. ( A ) Western blot analysis of cell lysates and immunoprecipitated samples show Flag-tagged RNase H1 and H2 expression from cells stably transformed with RNase H1 (H1) or H2 (H2) or wild type (control) HEK cell lines. ( B ) Co-selection of RNase H1 binding proteins by immunoprecipitation. Extracts from cells expressing the Flag-H1, Flag-H2, or HA-H1 cell lines were immunoprecipitated with either anti-Flag or anti-HA antibody. Co-precipitated proteins were resolved by SDS-PAGE, and visualized by silver staining. Protein bands that were different from the co-precipitated proteins from control cells were subjected to mass spectrometry. The protein bands corresponding to the tagged RNase H1, H2 and the co-precipitated P32 proteins are indicated. The size marker was SeeBlue Plus2 Pre-Stained Standard (Invitrogen). ( C ) 2D gel electrophoresis of proteins co-precipitated with Flag-H1 or Flag-H2. About 5 mg cell lysates were prepared for immunoprecipitation with anti-flag beads from cell lines which stably express Flag-H1 or Flag-H2. The immunoprecipitates were washed four times with RIPA buffer and directly sent to Applied Biomics Inc. (San Francisco, CA) for 2D gel electrophoresis coupled with MS analysis. In brief, the co-precipitated proteins from Flag-H1 or Flag-H2 cells were labeled by fluorescent DIGE CyDyers, respectively, followed by 2D gel electrophoresis. The protein image was scanned with a fluorescence detector. The figure illustrates the proteins differentially associated with RNase H1 (green) or H2 (red). The P32 protein was confirmed with mass spectrum from the extracted gel sample. Circled spots were identified as RNase H1, H2 or P32 by mass spectrometric analysis. ( D ) Both endogenous and expressed RNase H1 are co-precipitated with the expressed P32. Left panel: western blots with P32, RNase H1, or H2 antibodies for proteins co-precipitated using anti-HA antibody from extracts of control HeLa cells or cells transfected with HA-P32 expression plasmid. Right panel: western blots for proteins co-selected using anti-HA antibody from extracts of Flag-H1, Flag-H2 stable cell lines and control cells, all of which were transfected with HA-P32 expression plasmid. ( E ) Confirmation of the specific interaction between RNase H1 and P32. RNase H cleavage activity indicates that the P32 co-immunoprecipitated material contains only RNase H1 enzyme activity. Upper panel: Cleavage patterns of human RNase H1 and H2 from IP-coupled enzyme activity assays. Immunoprecipitations were performed with either anti-flag, anti-RNase H1 or anti-H2 antibodies from extracts of Flag-H1, Flag-H2 expressing cells or control cells. The co-precipitated samples were incubated for the indicated times with a 32 P-labeled RNA/DNA-methoxyethyl (MOE) gapmer duplex and the cleavage products were separated using denaturing gel electrophoresis. The preferred cleavage sites of RNase H1 and H2 are indicated with * or #, respectively. The positions of the preferred cleavage sites in the heteroduplex are shown in the middle panel with the sequences of the RNA substrate (upper strand) and the oligonucleotide (lower strand). The bold nucleotides in the oligonucleotide strand indicate the position of the MOE substitutions. Lower panel: only the RNase H1 enzyme activity was detected in the co-precipitated material from lysates containing tagged P32. Immunoprecipitations were performed with anti-HA antibody from extracts of Flag-H1 or Flag-H2 stable cell lines or control HEK cells, which were all transfected or not transfected with HA-P32 expression plasmid. The precipitated samples were analyzed for cleavage patterns as described above. The position of the cleavage bands relative to the sequence of the cleavage products is shown on the left. A partial alkaline digestion of the same labeled RNA was used as a sequence ladder. The cleavage pattern of purified human RNase H1 is shown at the far right of the lower panel.
    Figure Legend Snippet: Human RNase H1 is associated with P32. ( A ) Western blot analysis of cell lysates and immunoprecipitated samples show Flag-tagged RNase H1 and H2 expression from cells stably transformed with RNase H1 (H1) or H2 (H2) or wild type (control) HEK cell lines. ( B ) Co-selection of RNase H1 binding proteins by immunoprecipitation. Extracts from cells expressing the Flag-H1, Flag-H2, or HA-H1 cell lines were immunoprecipitated with either anti-Flag or anti-HA antibody. Co-precipitated proteins were resolved by SDS-PAGE, and visualized by silver staining. Protein bands that were different from the co-precipitated proteins from control cells were subjected to mass spectrometry. The protein bands corresponding to the tagged RNase H1, H2 and the co-precipitated P32 proteins are indicated. The size marker was SeeBlue Plus2 Pre-Stained Standard (Invitrogen). ( C ) 2D gel electrophoresis of proteins co-precipitated with Flag-H1 or Flag-H2. About 5 mg cell lysates were prepared for immunoprecipitation with anti-flag beads from cell lines which stably express Flag-H1 or Flag-H2. The immunoprecipitates were washed four times with RIPA buffer and directly sent to Applied Biomics Inc. (San Francisco, CA) for 2D gel electrophoresis coupled with MS analysis. In brief, the co-precipitated proteins from Flag-H1 or Flag-H2 cells were labeled by fluorescent DIGE CyDyers, respectively, followed by 2D gel electrophoresis. The protein image was scanned with a fluorescence detector. The figure illustrates the proteins differentially associated with RNase H1 (green) or H2 (red). The P32 protein was confirmed with mass spectrum from the extracted gel sample. Circled spots were identified as RNase H1, H2 or P32 by mass spectrometric analysis. ( D ) Both endogenous and expressed RNase H1 are co-precipitated with the expressed P32. Left panel: western blots with P32, RNase H1, or H2 antibodies for proteins co-precipitated using anti-HA antibody from extracts of control HeLa cells or cells transfected with HA-P32 expression plasmid. Right panel: western blots for proteins co-selected using anti-HA antibody from extracts of Flag-H1, Flag-H2 stable cell lines and control cells, all of which were transfected with HA-P32 expression plasmid. ( E ) Confirmation of the specific interaction between RNase H1 and P32. RNase H cleavage activity indicates that the P32 co-immunoprecipitated material contains only RNase H1 enzyme activity. Upper panel: Cleavage patterns of human RNase H1 and H2 from IP-coupled enzyme activity assays. Immunoprecipitations were performed with either anti-flag, anti-RNase H1 or anti-H2 antibodies from extracts of Flag-H1, Flag-H2 expressing cells or control cells. The co-precipitated samples were incubated for the indicated times with a 32 P-labeled RNA/DNA-methoxyethyl (MOE) gapmer duplex and the cleavage products were separated using denaturing gel electrophoresis. The preferred cleavage sites of RNase H1 and H2 are indicated with * or #, respectively. The positions of the preferred cleavage sites in the heteroduplex are shown in the middle panel with the sequences of the RNA substrate (upper strand) and the oligonucleotide (lower strand). The bold nucleotides in the oligonucleotide strand indicate the position of the MOE substitutions. Lower panel: only the RNase H1 enzyme activity was detected in the co-precipitated material from lysates containing tagged P32. Immunoprecipitations were performed with anti-HA antibody from extracts of Flag-H1 or Flag-H2 stable cell lines or control HEK cells, which were all transfected or not transfected with HA-P32 expression plasmid. The precipitated samples were analyzed for cleavage patterns as described above. The position of the cleavage bands relative to the sequence of the cleavage products is shown on the left. A partial alkaline digestion of the same labeled RNA was used as a sequence ladder. The cleavage pattern of purified human RNase H1 is shown at the far right of the lower panel.

    Techniques Used: Western Blot, Immunoprecipitation, Expressing, Stable Transfection, Transformation Assay, Selection, Binding Assay, SDS Page, Silver Staining, Mass Spectrometry, Marker, Staining, Two-Dimensional Gel Electrophoresis, Electrophoresis, Labeling, Fluorescence, Transfection, Plasmid Preparation, Activity Assay, Incubation, Nucleic Acid Electrophoresis, Sequencing, Purification

    23) Product Images from "NRDE-2, the human homolog of fission yeast Nrl1, prevents DNA damage accumulation in human cells"

    Article Title: NRDE-2, the human homolog of fission yeast Nrl1, prevents DNA damage accumulation in human cells

    Journal: RNA Biology

    doi: 10.1080/15476286.2018.1467180

    R-loop levels at RPL13A, BACT and EGR1 are not affected by NRDE-2 or Mtr4 KD. (a) DRIP assays were performed in HeLa cell after 72 h of siRNAs transfection. DRIP signal was also measured after RNase H1 treatment (right). (b) DRIP signal was normalized to the signal in non-transfected cell. Error bars represent the average of two different experiments. SD shown. (c) NRDE-2 and Mtr4 KDs confirmation by WB. None = no transfection, NC: siRNA control transfection. (D) NRDE-2 ChIP at various loci. ChIP assays were performed in HeLa cell after 72 h of siRNAs transfection. n = 2
    Figure Legend Snippet: R-loop levels at RPL13A, BACT and EGR1 are not affected by NRDE-2 or Mtr4 KD. (a) DRIP assays were performed in HeLa cell after 72 h of siRNAs transfection. DRIP signal was also measured after RNase H1 treatment (right). (b) DRIP signal was normalized to the signal in non-transfected cell. Error bars represent the average of two different experiments. SD shown. (c) NRDE-2 and Mtr4 KDs confirmation by WB. None = no transfection, NC: siRNA control transfection. (D) NRDE-2 ChIP at various loci. ChIP assays were performed in HeLa cell after 72 h of siRNAs transfection. n = 2

    Techniques Used: Transfection, Western Blot, Chromatin Immunoprecipitation

    DSBs induced by NRDE-2 or Mtr4 KDs are R-loop independent. (a) IF of γH2AX signal in NRDE-2 KDed HeLa cells for 72h and transfected (H1) or not (no H1) with GFP-RNase H1 for 48h. Signal quantifications are shown at the bottom. n = 189 cells, SE shown. (b) Quantification of γH2AX signal in Mtr4 KDed cells expressing GFP-RNase H1 (H1) or not (no H1). n = 78 cells, SE shown. White arrows show cells expressing GFP-RNase H1 and high level of γH2AX signal.
    Figure Legend Snippet: DSBs induced by NRDE-2 or Mtr4 KDs are R-loop independent. (a) IF of γH2AX signal in NRDE-2 KDed HeLa cells for 72h and transfected (H1) or not (no H1) with GFP-RNase H1 for 48h. Signal quantifications are shown at the bottom. n = 189 cells, SE shown. (b) Quantification of γH2AX signal in Mtr4 KDed cells expressing GFP-RNase H1 (H1) or not (no H1). n = 78 cells, SE shown. White arrows show cells expressing GFP-RNase H1 and high level of γH2AX signal.

    Techniques Used: Transfection, Expressing

    24) Product Images from "RNase HII saves rnhA mutant Escherichia coli from R-loop-associated chromosomal fragmentation"

    Article Title: RNase HII saves rnhA mutant Escherichia coli from R-loop-associated chromosomal fragmentation

    Journal: Journal of molecular biology

    doi: 10.1016/j.jmb.2017.08.004

    Chromosome fragmentation analysis by RNase HI, RNase HII and RNase A treatment in vitro A. A scheme of various hypothetical R-lesions (R-tract, two types of R-gaps) with positions of cleavage by RNase HI, HII and A (in low salt (LS) and high salt (HS) conditions) shown with arrows of the corresponding color. Small blue “d” letters, dNs; small orange “r” letters, rNs. The strand polarity in a duplex is identified on the left. B. A representative pulsed-field gel detecting chromosomal fragmentation after RNase HII treatment. The lanes are marked either with “b” (buffer treatment control) or “H2” (RNase HII treatment). Strains: WT, AB1157; rnhA , L-413; rnhB , L-415; rnhAB , L-416; uvrA rnhAB , L-417. C. Quantification of the RNase treatment-induced fragmentation. The plotted values are means ± SEM from 3-6 independent measurements from gels like in “B”. For RNase A treatment, both low salt (LS) and high salt (HS) conditions are plotted. Since individual fragmentation values are differences between the enzyme and the buffer treatments, some values are negative.
    Figure Legend Snippet: Chromosome fragmentation analysis by RNase HI, RNase HII and RNase A treatment in vitro A. A scheme of various hypothetical R-lesions (R-tract, two types of R-gaps) with positions of cleavage by RNase HI, HII and A (in low salt (LS) and high salt (HS) conditions) shown with arrows of the corresponding color. Small blue “d” letters, dNs; small orange “r” letters, rNs. The strand polarity in a duplex is identified on the left. B. A representative pulsed-field gel detecting chromosomal fragmentation after RNase HII treatment. The lanes are marked either with “b” (buffer treatment control) or “H2” (RNase HII treatment). Strains: WT, AB1157; rnhA , L-413; rnhB , L-415; rnhAB , L-416; uvrA rnhAB , L-417. C. Quantification of the RNase treatment-induced fragmentation. The plotted values are means ± SEM from 3-6 independent measurements from gels like in “B”. For RNase A treatment, both low salt (LS) and high salt (HS) conditions are plotted. Since individual fragmentation values are differences between the enzyme and the buffer treatments, some values are negative.

    Techniques Used: In Vitro, Pulsed-Field Gel

    Growth, morphology and viability of the double rnhAB mutants A. A scheme of in vivo substrates of the two RNase H enzymes. The common substrate, framed in bright green, is the RNA-run with at least four contiguous rNs, which we call “R-tract”. HI and H1, HII and H2 refer to RNase H enzymes of prokaryotes and eukaryotes accordingly. B. Colony size on LB agar, 37°C, 24 hours. Strains: WT, AB1157; Δ rnhA , L-413; Δ rnhB , L-415; Δ rnhAB , L-416. C. Images of rnh and wild type strains stained with DAPI and observed by Hiraga's fluo-phase combined method. Cells were grown at 37°C in LB. The strains are like in “B”. D. Viability of the strains, determined as the ratio of the colony forming units (CFUs) to the microscopic counts in the same volume of the culture. Overnight cultures grown at 30°C were diluted and grown at the temperature (indicated by the first number) to OD 0.2-0.3 (about 2 hours), then cultures were serially diluted and plated on LB plates developed for 16 hours at the temperature indicated by the second number in pairs. Average viability (± SEM) of the eight WT measurements and six measurements for the rnhAB mutant cells is shown (the low titers of the two MG1655 Δ rnhAB cultures at 42°C were not used in the calculation). Strains: AB1157, L-416, MG1655, L-419. E. An enlarged image of the rnhAB mutant cells (processed as in panel C), to show nucleoids of both filamenting and normal-looking cells in some detail. F. Anaerobic growth inhibition of the rnhA and anaerobic lethality of rnhAB strains. Dilution-spotting of strains (like in “B”) was done in an anaerobic chamber on LB plates. Plates were incubated at room temperature in the chamber for 24 hours, then shifted to 28°C aerobic conditions for another 48 hours. G. The uvrA defect further reduces the colony size of the rnhAB double mutant. Strains: rnhAB , L-416; uvrA rnhA , L-414; uvrA rnhAB , L-417.
    Figure Legend Snippet: Growth, morphology and viability of the double rnhAB mutants A. A scheme of in vivo substrates of the two RNase H enzymes. The common substrate, framed in bright green, is the RNA-run with at least four contiguous rNs, which we call “R-tract”. HI and H1, HII and H2 refer to RNase H enzymes of prokaryotes and eukaryotes accordingly. B. Colony size on LB agar, 37°C, 24 hours. Strains: WT, AB1157; Δ rnhA , L-413; Δ rnhB , L-415; Δ rnhAB , L-416. C. Images of rnh and wild type strains stained with DAPI and observed by Hiraga's fluo-phase combined method. Cells were grown at 37°C in LB. The strains are like in “B”. D. Viability of the strains, determined as the ratio of the colony forming units (CFUs) to the microscopic counts in the same volume of the culture. Overnight cultures grown at 30°C were diluted and grown at the temperature (indicated by the first number) to OD 0.2-0.3 (about 2 hours), then cultures were serially diluted and plated on LB plates developed for 16 hours at the temperature indicated by the second number in pairs. Average viability (± SEM) of the eight WT measurements and six measurements for the rnhAB mutant cells is shown (the low titers of the two MG1655 Δ rnhAB cultures at 42°C were not used in the calculation). Strains: AB1157, L-416, MG1655, L-419. E. An enlarged image of the rnhAB mutant cells (processed as in panel C), to show nucleoids of both filamenting and normal-looking cells in some detail. F. Anaerobic growth inhibition of the rnhA and anaerobic lethality of rnhAB strains. Dilution-spotting of strains (like in “B”) was done in an anaerobic chamber on LB plates. Plates were incubated at room temperature in the chamber for 24 hours, then shifted to 28°C aerobic conditions for another 48 hours. G. The uvrA defect further reduces the colony size of the rnhAB double mutant. Strains: rnhAB , L-416; uvrA rnhA , L-414; uvrA rnhAB , L-417.

    Techniques Used: In Vivo, Staining, Mutagenesis, Inhibition, Incubation

    Verification of RNase HI and RNase HII rN-DNA substrate specificity in vitro and the rN-density in DNA of the RNase H + cells and rnh mutants A. A scheme of the two double stranded oligo substrates: 38R1 (single rN) and 34R5 (five consecutive rN). The 32 P label at the 5′ end is shown as a red asterisk. DNA nucleotides are shown as blue lower case “d”, ribonucleotides are orange uppercase “R”. B. Products of the rN-DNA substrate hydrolysis by E. coli RNase HI and RNase HII enzymes. The radiolabelled rN-containing dsDNA oligos (shown in A) were incubated with the RNase HI or RNase HII enzymes. “0.1 M NaOH” and “Na Carb. pH 9.3” refer to alkali conditions in which rN hydrolysis produces reference size products. Numbers “1” or “5” refer to 38R1 or 34R5 oligos (A); ss/ds refers to whether the substrate used in the reaction was single-stranded or double-stranded. RNase H1 and RNase H2 were the E. coli enzymes RNase HI and RNase HII. RNase H1-1 and RNase H1-2 were RNase HI enzymes from different producers. The numbers on the side of the gel represent the sizes of the substrate and cleavage products. The reaction products were analyzed in 18% urea-PAGE gel. C. Only 34R5 oligo was used as either ss or ds substrate. All designations are like in “B”. D. Treatment with RNase HII of the plasmid isolated by alkaline lysis protocol. SCM, supercoiled monomer; b, buffer; H2, RNase HII. Plasmid: pEAK86, plasmid isolation was done at 0°C. Strains for results shown in panels D-I were: WT, AB1157; rnhA , L-413; rnhB , L-415; rnhAB , L-416; uvrA rnhAB L-417. Product of the reactions were run in 1.1% agarose gel; autoradiogram of the representative Southern blot with the radiolabelled pEAK86 DNA as a probe is shown here and also in E and G. E. Treatment with either RNase HI or RNase HII enzymes of the plasmid isolated by the total genomic DNA protocol. SC, supercoiled plasmid; relaxed, relaxed plasmid; chrom., chromosomal DNA. Plasmid: pEAK86. Analysis of plasmid species was carried out as in D. F. Summary of quantification of the RNaseHII-revealed density of rNs in plasmid DNA isolated by various methods from the rnhAB double mutant. The density calculations are described in Methods. “Form.”, formamide. G. Alkali treatment analysis of rN-density. The plasmid DNA isolated by alkaline lysis at 0°C, was linearized and treated with NaOH. Treatment: “—”, no treatment; 0°, 0.2 M NaOH, 20 mM EDTA treatment on ice for 20 min; 45°, 0.3 M NaOH, 20 mM EDTA treatment at 45°C for 90 minutes. ds, linearized plasmid DNA, ss -single stranded plasmid. The samples were run in 1.1% agarose in TAE buffer, at 4°C. H. Summary of quantification of the rN-density determined by either RNase HII or by alkali treatments (from gels like in “G”). Various mutant comparison data are shown, pEAK86 was purified by alkaline lysis only, values are means of three independent measurements ± SEM. The star identifies the value already reported in panel “F”. I. R-loop removal by RNase HI or by RNase A. pAM34 isolated from rnhA (strain L-413) by the total genomic DNA protocol.
    Figure Legend Snippet: Verification of RNase HI and RNase HII rN-DNA substrate specificity in vitro and the rN-density in DNA of the RNase H + cells and rnh mutants A. A scheme of the two double stranded oligo substrates: 38R1 (single rN) and 34R5 (five consecutive rN). The 32 P label at the 5′ end is shown as a red asterisk. DNA nucleotides are shown as blue lower case “d”, ribonucleotides are orange uppercase “R”. B. Products of the rN-DNA substrate hydrolysis by E. coli RNase HI and RNase HII enzymes. The radiolabelled rN-containing dsDNA oligos (shown in A) were incubated with the RNase HI or RNase HII enzymes. “0.1 M NaOH” and “Na Carb. pH 9.3” refer to alkali conditions in which rN hydrolysis produces reference size products. Numbers “1” or “5” refer to 38R1 or 34R5 oligos (A); ss/ds refers to whether the substrate used in the reaction was single-stranded or double-stranded. RNase H1 and RNase H2 were the E. coli enzymes RNase HI and RNase HII. RNase H1-1 and RNase H1-2 were RNase HI enzymes from different producers. The numbers on the side of the gel represent the sizes of the substrate and cleavage products. The reaction products were analyzed in 18% urea-PAGE gel. C. Only 34R5 oligo was used as either ss or ds substrate. All designations are like in “B”. D. Treatment with RNase HII of the plasmid isolated by alkaline lysis protocol. SCM, supercoiled monomer; b, buffer; H2, RNase HII. Plasmid: pEAK86, plasmid isolation was done at 0°C. Strains for results shown in panels D-I were: WT, AB1157; rnhA , L-413; rnhB , L-415; rnhAB , L-416; uvrA rnhAB L-417. Product of the reactions were run in 1.1% agarose gel; autoradiogram of the representative Southern blot with the radiolabelled pEAK86 DNA as a probe is shown here and also in E and G. E. Treatment with either RNase HI or RNase HII enzymes of the plasmid isolated by the total genomic DNA protocol. SC, supercoiled plasmid; relaxed, relaxed plasmid; chrom., chromosomal DNA. Plasmid: pEAK86. Analysis of plasmid species was carried out as in D. F. Summary of quantification of the RNaseHII-revealed density of rNs in plasmid DNA isolated by various methods from the rnhAB double mutant. The density calculations are described in Methods. “Form.”, formamide. G. Alkali treatment analysis of rN-density. The plasmid DNA isolated by alkaline lysis at 0°C, was linearized and treated with NaOH. Treatment: “—”, no treatment; 0°, 0.2 M NaOH, 20 mM EDTA treatment on ice for 20 min; 45°, 0.3 M NaOH, 20 mM EDTA treatment at 45°C for 90 minutes. ds, linearized plasmid DNA, ss -single stranded plasmid. The samples were run in 1.1% agarose in TAE buffer, at 4°C. H. Summary of quantification of the rN-density determined by either RNase HII or by alkali treatments (from gels like in “G”). Various mutant comparison data are shown, pEAK86 was purified by alkaline lysis only, values are means of three independent measurements ± SEM. The star identifies the value already reported in panel “F”. I. R-loop removal by RNase HI or by RNase A. pAM34 isolated from rnhA (strain L-413) by the total genomic DNA protocol.

    Techniques Used: In Vitro, Incubation, Polyacrylamide Gel Electrophoresis, Plasmid Preparation, Isolation, Alkaline Lysis, Agarose Gel Electrophoresis, Southern Blot, Mutagenesis, Purification

    25) Product Images from "Identification and characterization of intracellular proteins that bind oligonucleotides with phosphorothioate linkages"

    Article Title: Identification and characterization of intracellular proteins that bind oligonucleotides with phosphorothioate linkages

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv143

    Ku80 and P54nrb compete with RNase H1 for binding to ASO/RNA duplex. ( A ) Reduction of P54nrb or Ku80 protein levels by siRNA treatment increased the binding of RNase H1 protein to the ASO/RNA-like duplex, as determined by affinity selection using an ASO/RNA-like duplex, followed by western analyses. ( B ) Simultaneous treatment with siRNAs targeting P54nrb and Ku80 reduced levels of both proteins, as shown by western analysis. ( C ) Simultaneous reduction of P54nrb and Ku80 led to a significant increase in the binding of RNase H1 to the ASO/RNA-like duplex. Silver staining of an aliquot of the affinity selected proteins analyzed on a separate SDS-PAGE is shown as a loading control. ( D ) The protein levels of RNase H1, Ku80 and P54nrb in control cells (UTC) or cells over-expressing RNase H1 were evaluated by western blot. ( E ) Over-expression of RNase H1 led to reduced binding of P54nrb and Ku80 proteins to the ASO/RNA-like duplex, as determined by affinity selection followed by western analyses. The numbers below the lanes indicate the estimated protein level relative to control.
    Figure Legend Snippet: Ku80 and P54nrb compete with RNase H1 for binding to ASO/RNA duplex. ( A ) Reduction of P54nrb or Ku80 protein levels by siRNA treatment increased the binding of RNase H1 protein to the ASO/RNA-like duplex, as determined by affinity selection using an ASO/RNA-like duplex, followed by western analyses. ( B ) Simultaneous treatment with siRNAs targeting P54nrb and Ku80 reduced levels of both proteins, as shown by western analysis. ( C ) Simultaneous reduction of P54nrb and Ku80 led to a significant increase in the binding of RNase H1 to the ASO/RNA-like duplex. Silver staining of an aliquot of the affinity selected proteins analyzed on a separate SDS-PAGE is shown as a loading control. ( D ) The protein levels of RNase H1, Ku80 and P54nrb in control cells (UTC) or cells over-expressing RNase H1 were evaluated by western blot. ( E ) Over-expression of RNase H1 led to reduced binding of P54nrb and Ku80 proteins to the ASO/RNA-like duplex, as determined by affinity selection followed by western analyses. The numbers below the lanes indicate the estimated protein level relative to control.

    Techniques Used: Binding Assay, Allele-specific Oligonucleotide, Selection, Western Blot, Silver Staining, SDS Page, Expressing, Over Expression

    26) Product Images from "An Upstream Open Reading Frame and the Context of the Two AUG Codons Affect the Abundance of Mitochondrial and Nuclear RNase H1 ▿An Upstream Open Reading Frame and the Context of the Two AUG Codons Affect the Abundance of Mitochondrial and Nuclear RNase H1 ▿ †"

    Article Title: An Upstream Open Reading Frame and the Context of the Two AUG Codons Affect the Abundance of Mitochondrial and Nuclear RNase H1 ▿An Upstream Open Reading Frame and the Context of the Two AUG Codons Affect the Abundance of Mitochondrial and Nuclear RNase H1 ▿ †

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00619-10

    Overexpression of RNase H1 in mitochondria impairs growth and results in mtDNA loss. (A) FT293-CAT, FT293-M27, FT293-M1-M27I, and FT293-M1 cells were grown in the media with (filled squares) or without (open squares) 1 μg/ml doxycycline. The FT293-CAT cell line has the CAT gene inserted at the flip-in site. Likewise, the other FT293 cell lines have the cDNA for the Rnaseh1 mRNA, starting at M1 (FT293-M1), M27 (FT293-M27), or M1 with the M27 AUG changed to AUC (FT293-M1-M27I). The numbers of cells were counted with a hemocytometer. (B) RNase H1-HA-NESi is expressed, activated, and localized to mitochondria in 293 cells. RNase H1 mRNA levels were assayed by RT-qPCR at different time points and dosages of doxycycline. The initial black bars are the parental FT293 cell line lacking RNase H1-HA-NESi. Subsequent bars from left to right in each set are cells with RNase H-HA-NESi expressed at 0 (red), 1 (blue), 3 (yellow), and 10 (green) ng/ml doxycycline. Protein contents of cell lysates were analyzed by Coomassie brilliant blue (CBB) stain, Western blotting for the HA epitope, and the RNase H1 gel activity assay with poly(rA)/poly(dT) as the substrate. Equal amounts of total protein (as assessed from the CBB staining) were loaded in each lane. For clarity, activity is shown as a negative image. Expression of RNase H1-HA-NESi was induced for 6 days using 0, 3, 30, and 60 ng/ml doxycycline. At the bottom and to the right is shown confocal microscopy of FT293-RNase H1-HA-NESi. After 24 h of induction using 100 ng/ml doxycycline, the entire signal detected with the anti-HA antibody (green) colocalized with mitochondria (MitoTracker orange). mtDNA copy number decreases as the dosage of doxycycline and days of incubation increase. For each day, the first bar (black) is FT293 without the cDNA for human RNase H1 treated with 10 ng/ml doxycycline. Subsequent bars from left to right in each set contain 0 (violet), 0.1 (red), 1 (blue), 3 (yellow), or 10 (green) ng/ml doxycycline. At day 6, the values for 1, 3, and 10 ng/ml doxycycline are 0.036, 0.032, and 0.027, respectively. At day 9, the values for 1, 3, and 10 ng/ml doxycycline are 0.018, 0.011, and 0.010, respectively.
    Figure Legend Snippet: Overexpression of RNase H1 in mitochondria impairs growth and results in mtDNA loss. (A) FT293-CAT, FT293-M27, FT293-M1-M27I, and FT293-M1 cells were grown in the media with (filled squares) or without (open squares) 1 μg/ml doxycycline. The FT293-CAT cell line has the CAT gene inserted at the flip-in site. Likewise, the other FT293 cell lines have the cDNA for the Rnaseh1 mRNA, starting at M1 (FT293-M1), M27 (FT293-M27), or M1 with the M27 AUG changed to AUC (FT293-M1-M27I). The numbers of cells were counted with a hemocytometer. (B) RNase H1-HA-NESi is expressed, activated, and localized to mitochondria in 293 cells. RNase H1 mRNA levels were assayed by RT-qPCR at different time points and dosages of doxycycline. The initial black bars are the parental FT293 cell line lacking RNase H1-HA-NESi. Subsequent bars from left to right in each set are cells with RNase H-HA-NESi expressed at 0 (red), 1 (blue), 3 (yellow), and 10 (green) ng/ml doxycycline. Protein contents of cell lysates were analyzed by Coomassie brilliant blue (CBB) stain, Western blotting for the HA epitope, and the RNase H1 gel activity assay with poly(rA)/poly(dT) as the substrate. Equal amounts of total protein (as assessed from the CBB staining) were loaded in each lane. For clarity, activity is shown as a negative image. Expression of RNase H1-HA-NESi was induced for 6 days using 0, 3, 30, and 60 ng/ml doxycycline. At the bottom and to the right is shown confocal microscopy of FT293-RNase H1-HA-NESi. After 24 h of induction using 100 ng/ml doxycycline, the entire signal detected with the anti-HA antibody (green) colocalized with mitochondria (MitoTracker orange). mtDNA copy number decreases as the dosage of doxycycline and days of incubation increase. For each day, the first bar (black) is FT293 without the cDNA for human RNase H1 treated with 10 ng/ml doxycycline. Subsequent bars from left to right in each set contain 0 (violet), 0.1 (red), 1 (blue), 3 (yellow), or 10 (green) ng/ml doxycycline. At day 6, the values for 1, 3, and 10 ng/ml doxycycline are 0.036, 0.032, and 0.027, respectively. At day 9, the values for 1, 3, and 10 ng/ml doxycycline are 0.018, 0.011, and 0.010, respectively.

    Techniques Used: Over Expression, Quantitative RT-PCR, Staining, Western Blot, Activity Assay, Expressing, Confocal Microscopy, Incubation

    27) Product Images from "Regulation of ATR activity via the RNA polymerase II associated factors CDC73 and PNUTS-PP1"

    Article Title: Regulation of ATR activity via the RNA polymerase II associated factors CDC73 and PNUTS-PP1

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1233

    CDC73 is required for high ATR signaling in S-phase and activation of the endogenous G2 checkpoint after PNUTS depletion, and interacts with ATR and RNAPII. ( A ) Flow cytometry charts showing γH2AX staining versus DNA content as in 3A) of scr, siPNUTS or siPNUTS and siCDC73 transfected cells harvested at 72 h after siRNA transfection with and without 1 h treatment with VE-822. Quantifications show relative median γH2AX levels in indicated S-phase cells (black box). ( n = 3) * P
    Figure Legend Snippet: CDC73 is required for high ATR signaling in S-phase and activation of the endogenous G2 checkpoint after PNUTS depletion, and interacts with ATR and RNAPII. ( A ) Flow cytometry charts showing γH2AX staining versus DNA content as in 3A) of scr, siPNUTS or siPNUTS and siCDC73 transfected cells harvested at 72 h after siRNA transfection with and without 1 h treatment with VE-822. Quantifications show relative median γH2AX levels in indicated S-phase cells (black box). ( n = 3) * P

    Techniques Used: Activation Assay, Flow Cytometry, Cytometry, Staining, Transfection

    28) Product Images from "BRCA2 controls DNA:RNA hybrid level at DSBs by mediating RNase H2 recruitment"

    Article Title: BRCA2 controls DNA:RNA hybrid level at DSBs by mediating RNase H2 recruitment

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07799-2

    DNA:RNA hybrids are directly recognized by BRCA1 in vitro and in vivo. a Representative pictures of super-resolution imaging analysis of BRCA1 (cyan) and DNA:RNA hybrids (yellow) colocalization in S-phase synchronized NCS-treated U2OS cells. Scale bar: 5 μm. b Dot plot shows the normalized number of overlaps relative to random of BRCA1 and DNA:RNA hybrids signals in S-phase U2OS cells treated with DSMO or NCS. At least n = 40 events were counted from three independent experiments. Lines represent mean ± s.e.m. c Electrophoretic mobility shift assay (EMSA) of purified recombinant human BRCA1 or BRCA1-BARD1 with end-labeled (*) double-stranded DNA or DNA:RNA substrates. d Graph showing the percentage of protein-bound substrate at respective protein concentrations. Error bars represent s.e.m. ( n = 2 independent experiments). e Representative images of BRCA1 foci co-stained with cyclin A, as S/G2-phase marker, in irradiated (2 Gy) U2OS cells over-expressing GFP or GFP-RNase H1 (GFP-RH1). Scale bar: 5 μm. f Dot plot shows the number of foci in e . At least n = 80 cells were counted from at least three independent experiments. Lines represent mean ± s.e.m. g Representative images of BRCA1 foci co-stained with cyclin A, as S/G2-phase marker, in irradiated (2 Gy) U2OS cells treated with RNase H prior to fixation. Scale bar: 10 μm. h Dot plot shows the number of foci in g . At least n = 80 cells were counted from three independent experiments. Lines represent mean ± s.e.m. * P
    Figure Legend Snippet: DNA:RNA hybrids are directly recognized by BRCA1 in vitro and in vivo. a Representative pictures of super-resolution imaging analysis of BRCA1 (cyan) and DNA:RNA hybrids (yellow) colocalization in S-phase synchronized NCS-treated U2OS cells. Scale bar: 5 μm. b Dot plot shows the normalized number of overlaps relative to random of BRCA1 and DNA:RNA hybrids signals in S-phase U2OS cells treated with DSMO or NCS. At least n = 40 events were counted from three independent experiments. Lines represent mean ± s.e.m. c Electrophoretic mobility shift assay (EMSA) of purified recombinant human BRCA1 or BRCA1-BARD1 with end-labeled (*) double-stranded DNA or DNA:RNA substrates. d Graph showing the percentage of protein-bound substrate at respective protein concentrations. Error bars represent s.e.m. ( n = 2 independent experiments). e Representative images of BRCA1 foci co-stained with cyclin A, as S/G2-phase marker, in irradiated (2 Gy) U2OS cells over-expressing GFP or GFP-RNase H1 (GFP-RH1). Scale bar: 5 μm. f Dot plot shows the number of foci in e . At least n = 80 cells were counted from at least three independent experiments. Lines represent mean ± s.e.m. g Representative images of BRCA1 foci co-stained with cyclin A, as S/G2-phase marker, in irradiated (2 Gy) U2OS cells treated with RNase H prior to fixation. Scale bar: 10 μm. h Dot plot shows the number of foci in g . At least n = 80 cells were counted from three independent experiments. Lines represent mean ± s.e.m. * P

    Techniques Used: In Vitro, In Vivo, Imaging, Electrophoretic Mobility Shift Assay, Purification, Recombinant, Labeling, Staining, Marker, Irradiation, Expressing

    29) Product Images from "The RNA binding protein Npl3 promotes resection of DNA double-strand breaks by regulating the levels of Exo1"

    Article Title: The RNA binding protein Npl3 promotes resection of DNA double-strand breaks by regulating the levels of Exo1

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx347

    EXO1 RNA in the absence of Npl3. ( A ) Schematic representation of the EXO1 locus. Primer pairs (PP1-PP6) used for qRT-PCR are indicated by arrows. A bar indicates the 1437 bp-DNA probe internal to the EXO1 coding sequence (+628 to + 2065 from the ATG initiation codon) used for northern blot. ( B ) Total RNA was extracted from exponentially growing YEPD cell cultures of the indicated strains and subjected to quantitative reverse transcriptase PCR (qRT-PCR) with primer pairs located into the EXO1 (PP1 in (A)) and ALG9 coding sequences. The EXO1 RNA levels relative to wild type (set to 1.0) were calculated using ΔΔCt method after normalization to the ALG9 RNA levels for each sample. The mean values are represented with error bars denoting SD ( n = 5). ( C–E ) Total RNA extracted from the indicated cell cultures was subjected to northern blot and hybridized with the probe as in (A). The agarose gels were stained with ethidium bromide to detect 18S and 25S rRNAs (bottom). ( F ) Total RNA extracted from wild type and npl3Δ cells was subjected to 5΄ RACE to visualize the EXO1 5΄ partial cDNA ends. After reverse transcription with a EXO1 specific primer and poly(A) tailing, two subsequent PCR reactions were performed with primers annealing to the appended tail and to the EXO1 coding sequence 718 and 248 bp downstream the EXO1 initiation codon. The final PCR products were separated on a 1.5% agarose gel and visualized with ethidium bromide. ( G ) Total RNA as in (F) was subjected to qRT-PCR with primer pairs depicted in (A), or located in the ALG9 coding sequence. The amount of products obtained with different EXO1 primer pairs was normalized to the ALG9 product using ΔΔCt method. Then, the normalized RNA levels estimated with the different primer pairs in the EXO1 locus were normalized to the RNA levels evaluated with the PP1 primer pair and set to 1.0 for each strain. The mean values are represented with error bars denoting SD ( n = 4).
    Figure Legend Snippet: EXO1 RNA in the absence of Npl3. ( A ) Schematic representation of the EXO1 locus. Primer pairs (PP1-PP6) used for qRT-PCR are indicated by arrows. A bar indicates the 1437 bp-DNA probe internal to the EXO1 coding sequence (+628 to + 2065 from the ATG initiation codon) used for northern blot. ( B ) Total RNA was extracted from exponentially growing YEPD cell cultures of the indicated strains and subjected to quantitative reverse transcriptase PCR (qRT-PCR) with primer pairs located into the EXO1 (PP1 in (A)) and ALG9 coding sequences. The EXO1 RNA levels relative to wild type (set to 1.0) were calculated using ΔΔCt method after normalization to the ALG9 RNA levels for each sample. The mean values are represented with error bars denoting SD ( n = 5). ( C–E ) Total RNA extracted from the indicated cell cultures was subjected to northern blot and hybridized with the probe as in (A). The agarose gels were stained with ethidium bromide to detect 18S and 25S rRNAs (bottom). ( F ) Total RNA extracted from wild type and npl3Δ cells was subjected to 5΄ RACE to visualize the EXO1 5΄ partial cDNA ends. After reverse transcription with a EXO1 specific primer and poly(A) tailing, two subsequent PCR reactions were performed with primers annealing to the appended tail and to the EXO1 coding sequence 718 and 248 bp downstream the EXO1 initiation codon. The final PCR products were separated on a 1.5% agarose gel and visualized with ethidium bromide. ( G ) Total RNA as in (F) was subjected to qRT-PCR with primer pairs depicted in (A), or located in the ALG9 coding sequence. The amount of products obtained with different EXO1 primer pairs was normalized to the ALG9 product using ΔΔCt method. Then, the normalized RNA levels estimated with the different primer pairs in the EXO1 locus were normalized to the RNA levels evaluated with the PP1 primer pair and set to 1.0 for each strain. The mean values are represented with error bars denoting SD ( n = 4).

    Techniques Used: Quantitative RT-PCR, Sequencing, Northern Blot, Polymerase Chain Reaction, Staining, Agarose Gel Electrophoresis

    30) Product Images from "Genome Instability and Transcription Elongation Impairment in Human Cells Depleted of THO/TREX"

    Article Title: Genome Instability and Transcription Elongation Impairment in Human Cells Depleted of THO/TREX

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1002386

    Recombination is increased after THOC1 depletion. A) Scheme of the pIREC direct-repeat recombination construct used to generate stable HeLa cell lines. B) Recombination analysis using the HeRG stable cell line depleted of THOC1 by siRNA. C) Recombination analysis in the HeTH-4 stable cell lines (−DOX and +DOX) transfected with the plasmid pIREC-direct repeat containing vector. The recombination frequency was measured by FACs analysis as GFP positive cells, 96 h after transfection (B and C). Average and SE from three independent experiments are shown. When the P value of the difference with the siC control calculated with the Mann Whitney test is
    Figure Legend Snippet: Recombination is increased after THOC1 depletion. A) Scheme of the pIREC direct-repeat recombination construct used to generate stable HeLa cell lines. B) Recombination analysis using the HeRG stable cell line depleted of THOC1 by siRNA. C) Recombination analysis in the HeTH-4 stable cell lines (−DOX and +DOX) transfected with the plasmid pIREC-direct repeat containing vector. The recombination frequency was measured by FACs analysis as GFP positive cells, 96 h after transfection (B and C). Average and SE from three independent experiments are shown. When the P value of the difference with the siC control calculated with the Mann Whitney test is

    Techniques Used: Construct, Stable Transfection, Transfection, Plasmid Preparation, FACS, MANN-WHITNEY

    31) Product Images from "The core spliceosome as target and effector of non-canonical ATM signaling"

    Article Title: The core spliceosome as target and effector of non-canonical ATM signaling

    Journal: Nature

    doi: 10.1038/nature14512

    Reciprocal regulation between spliceosome mobilization and R loop-dependent ATM signaling a, Immunofluorescence of ATM activation in quiescent HDFs. b, Recruitment of RNAseH1(D145N)-GFP and mCherry-XPA at UV-C microirradiation sites ( n=10 , mean ± s.e.m., T-test). c,d,e,f FRAP showing SNRNP40-GFP mobilization in (c) untransfected and mCherry-RNaseH1 expressing U2OS cells, (d) after RNAseH1/H2A silencing, (e) in quiescent HDFs treated with DRB and/or IR and (f) after UV or CPT treatment. ( n=30 , mean ± s.e.m., one-way ANOVA). g,h, Intron retention assayed by RT-PCR in quiescent cells after (f) silencing of RNaseH1/H2A or (g) combined IR/DRB treatments ( n=2 , mean ± s.d., one-way ANOVA). (i) Model of UV-triggered and R-loop/ATM-augmented spliceosome mobilization.
    Figure Legend Snippet: Reciprocal regulation between spliceosome mobilization and R loop-dependent ATM signaling a, Immunofluorescence of ATM activation in quiescent HDFs. b, Recruitment of RNAseH1(D145N)-GFP and mCherry-XPA at UV-C microirradiation sites ( n=10 , mean ± s.e.m., T-test). c,d,e,f FRAP showing SNRNP40-GFP mobilization in (c) untransfected and mCherry-RNaseH1 expressing U2OS cells, (d) after RNAseH1/H2A silencing, (e) in quiescent HDFs treated with DRB and/or IR and (f) after UV or CPT treatment. ( n=30 , mean ± s.e.m., one-way ANOVA). g,h, Intron retention assayed by RT-PCR in quiescent cells after (f) silencing of RNaseH1/H2A or (g) combined IR/DRB treatments ( n=2 , mean ± s.d., one-way ANOVA). (i) Model of UV-triggered and R-loop/ATM-augmented spliceosome mobilization.

    Techniques Used: Immunofluorescence, Activation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction

    Displacement of mature spliceosomes from subnuclear sites of UV-inflicted DNA damage a. U2Os cells stably expressing GFP-tagged SFs were UV irradiated (60 J/m 2 ) through isopore membranes resulting in DNA-lesion formation in small subnuclear areas. DNA damage sites (circled) were visualized by immunofluorescence using an antibody against the NER recognition factor XPC. Scale bar: 5 μm. b. SF3a1-GFP and PRP8-GFP depletion from UV-C laser micro-irradiation sites. Quantification of 20 cells from two independent experiments. eGFP localization at sites of DNA damage is used to demonstrate that depletion of eGFP-tagged SFs is not caused by photobleaching. c. UV-C laser micro-irradiation results in preferential displacement of U2 and U5-associated SFs from DNA damage sites. Quiescent HDFs were irradiated in an ≈1μm diameter nuclear area via a UV-C laser. GFP signal intensity, reflecting the abundance of GFP-tagged U1, U2, U4 and U5 snRNP components at UV-C DNA-damage sites, was quantified in the irradiated and in an unirradiated nuclear area (undamaged control). Plotted is the fluorescence signal intenisty expressed as % of that prior to irradiation, at the 1 min. time point. Cells expressing free eGFP were used as negative control. Representative from three independent experiment ( n=12 , mean ± s.e.m., paired T-test). d . Depletion of SFs from UV-C laser irradiation sites depends on active transcription. Transcription initiation was inhibited in quiescent HDFs by prolonged α-amanitin treatment (10 μM, ≥24h) prior to subnuclear UV-C laser irradiation. Plotted is the SNRNP40-GFP abundance in irradiated and unirradiated nuclear areas at 1 min post-irradiation. Represenative from three independent experiments ( n=12 , mean ± s.e.m., one-way ANOVA / Bonferroni).
    Figure Legend Snippet: Displacement of mature spliceosomes from subnuclear sites of UV-inflicted DNA damage a. U2Os cells stably expressing GFP-tagged SFs were UV irradiated (60 J/m 2 ) through isopore membranes resulting in DNA-lesion formation in small subnuclear areas. DNA damage sites (circled) were visualized by immunofluorescence using an antibody against the NER recognition factor XPC. Scale bar: 5 μm. b. SF3a1-GFP and PRP8-GFP depletion from UV-C laser micro-irradiation sites. Quantification of 20 cells from two independent experiments. eGFP localization at sites of DNA damage is used to demonstrate that depletion of eGFP-tagged SFs is not caused by photobleaching. c. UV-C laser micro-irradiation results in preferential displacement of U2 and U5-associated SFs from DNA damage sites. Quiescent HDFs were irradiated in an ≈1μm diameter nuclear area via a UV-C laser. GFP signal intensity, reflecting the abundance of GFP-tagged U1, U2, U4 and U5 snRNP components at UV-C DNA-damage sites, was quantified in the irradiated and in an unirradiated nuclear area (undamaged control). Plotted is the fluorescence signal intenisty expressed as % of that prior to irradiation, at the 1 min. time point. Cells expressing free eGFP were used as negative control. Representative from three independent experiment ( n=12 , mean ± s.e.m., paired T-test). d . Depletion of SFs from UV-C laser irradiation sites depends on active transcription. Transcription initiation was inhibited in quiescent HDFs by prolonged α-amanitin treatment (10 μM, ≥24h) prior to subnuclear UV-C laser irradiation. Plotted is the SNRNP40-GFP abundance in irradiated and unirradiated nuclear areas at 1 min post-irradiation. Represenative from three independent experiments ( n=12 , mean ± s.e.m., one-way ANOVA / Bonferroni).

    Techniques Used: Stable Transfection, Expressing, Irradiation, Immunofluorescence, Fluorescence, Negative Control

    a. Recruitment of GFP-RNaseH1(D145N) at local DNA-damage sites depends on endogenous levels of RNaseH activity. DNA damage was inflicted via a UV-C laser in ≈1 μm-diameter subnuclear areas of cells after silencing of RNaseH2A or overexpression of RNaseH1-mCherry. Recruitment of RNaseH1(D145N)-GFP at the irradiated sites was monitored by live-cell imaging. Plotted is the fluorescence intensity of RNaseH1(D145N)-GFP at 1 min. post irradiation, at the irradiated and in a non-irradiated nuclear area. Representative from three independent experiments ( n=10 , mean ± s.e.m., one-way ANOVA / Bonferroni). b and c. R-loop formation at sites of local UV-C laser irradiation. Immunofluorescence detection of R-Loops using the DNA:RNA hybrid-specific S9.6 antibody. Sites of irradiation are visualized by XPC immunodetection. (b) Dashed boxes indicate the magnified areas shown in the right panels. The dashed lines indicate the line-scan track used to quantify fluorescence intensity of S9.6 and anti-XPC (shown in in the graph). (c) Specificity of the antibody was confirmed by its increased sensitivity after RNase H2A silencing and its ability to detect R-loops when suboptimal doses of UV-C irradiation were applied. d. RNaseH1 accumulation at local DNA-damage sites depends on active transcription but not ATM activity. Transcription initiation was inhibited in quiescent HDFs by α-amanitin (10 μg/ml, 24h) prior to local UV-C laser irradiation. Plotted is the fluorescence intensity at 1 min. post irradiation of RNaseH1(D145N)-GFP at the irradiated and in a non-irradiated nuclear area for untreated, ATM-inhibitor and α-amanitin treated cells. Representative from three experiments ( n=10 , mean ± s.e.m., one-way ANOVA / Bonferroni). e. RNaseH1 overexpression inhibits the UV-dependent spliceosome mobilization. FRAP of U2Os cells stably expressing GFP-tagged SF3a1 and PRP8 and transiently transfected with RNaseH1-mcherry. f. RNaseH1 and H2A silencing potentiates the UV-dependent spliceosome mobilization. RNaseH1 and H2 were silenced in U2Os cells expressing SF3a1-GFP or PRP8-GFP and SF mobility was assayed by FRAP. g. FRAP of SNRNP40-GFP in quiescent HDFs after RNaseH1/H2 silencing. e, f, g, n=30 , mean ± s.e.m., one-way ANOVA / Bonferroni.
    Figure Legend Snippet: a. Recruitment of GFP-RNaseH1(D145N) at local DNA-damage sites depends on endogenous levels of RNaseH activity. DNA damage was inflicted via a UV-C laser in ≈1 μm-diameter subnuclear areas of cells after silencing of RNaseH2A or overexpression of RNaseH1-mCherry. Recruitment of RNaseH1(D145N)-GFP at the irradiated sites was monitored by live-cell imaging. Plotted is the fluorescence intensity of RNaseH1(D145N)-GFP at 1 min. post irradiation, at the irradiated and in a non-irradiated nuclear area. Representative from three independent experiments ( n=10 , mean ± s.e.m., one-way ANOVA / Bonferroni). b and c. R-loop formation at sites of local UV-C laser irradiation. Immunofluorescence detection of R-Loops using the DNA:RNA hybrid-specific S9.6 antibody. Sites of irradiation are visualized by XPC immunodetection. (b) Dashed boxes indicate the magnified areas shown in the right panels. The dashed lines indicate the line-scan track used to quantify fluorescence intensity of S9.6 and anti-XPC (shown in in the graph). (c) Specificity of the antibody was confirmed by its increased sensitivity after RNase H2A silencing and its ability to detect R-loops when suboptimal doses of UV-C irradiation were applied. d. RNaseH1 accumulation at local DNA-damage sites depends on active transcription but not ATM activity. Transcription initiation was inhibited in quiescent HDFs by α-amanitin (10 μg/ml, 24h) prior to local UV-C laser irradiation. Plotted is the fluorescence intensity at 1 min. post irradiation of RNaseH1(D145N)-GFP at the irradiated and in a non-irradiated nuclear area for untreated, ATM-inhibitor and α-amanitin treated cells. Representative from three experiments ( n=10 , mean ± s.e.m., one-way ANOVA / Bonferroni). e. RNaseH1 overexpression inhibits the UV-dependent spliceosome mobilization. FRAP of U2Os cells stably expressing GFP-tagged SF3a1 and PRP8 and transiently transfected with RNaseH1-mcherry. f. RNaseH1 and H2A silencing potentiates the UV-dependent spliceosome mobilization. RNaseH1 and H2 were silenced in U2Os cells expressing SF3a1-GFP or PRP8-GFP and SF mobility was assayed by FRAP. g. FRAP of SNRNP40-GFP in quiescent HDFs after RNaseH1/H2 silencing. e, f, g, n=30 , mean ± s.e.m., one-way ANOVA / Bonferroni.

    Techniques Used: Activity Assay, Over Expression, Irradiation, Live Cell Imaging, Fluorescence, Immunofluorescence, Immunodetection, Stable Transfection, Expressing, Transfection

    Mobilization and displacement of mature spliceosomes from UV-C induced DNA damage sites a, Immunofluorescence detection of SNRNP40 and CPDs in U2Os cells UV-irradiated through porous membranes. b, SNRNP40-GFP depletion from UV-C laser microirradiation sites in U2Os cells; typical image (top) and fluoresence quantification of 20 cells (bottom). c, FRAP of UV-triggered SNRNP40-GFP mobilization in U2Os and quiescent HDFs ( n=25 ). d, FRAP of free eGFP or GFP-tagged SFs in UV-irradiated quiescent HDFs. Δ[ mobility ] = (Fluorescence irradiated – fluorescence non-irradiated cells) at 1 min post-bleaching ( n=25 , mean ± s.e.m., T-test and one-way ANOVA).
    Figure Legend Snippet: Mobilization and displacement of mature spliceosomes from UV-C induced DNA damage sites a, Immunofluorescence detection of SNRNP40 and CPDs in U2Os cells UV-irradiated through porous membranes. b, SNRNP40-GFP depletion from UV-C laser microirradiation sites in U2Os cells; typical image (top) and fluoresence quantification of 20 cells (bottom). c, FRAP of UV-triggered SNRNP40-GFP mobilization in U2Os and quiescent HDFs ( n=25 ). d, FRAP of free eGFP or GFP-tagged SFs in UV-irradiated quiescent HDFs. Δ[ mobility ] = (Fluorescence irradiated – fluorescence non-irradiated cells) at 1 min post-bleaching ( n=25 , mean ± s.e.m., T-test and one-way ANOVA).

    Techniques Used: Immunofluorescence, Irradiation, Fluorescence

    a. Schematic overview of the proteomic experiments for the identification of proteins that display UV-dependant chromatin association. b. Schematic outline of cell fractionation. c. Validation of chromatin isolation protocol for NER proteins that are reqruited to chromatin in response to DNA damage. Mock-treated or UV irradiated quiescent HDFs (20 J/m 2 , 1hr post-irradiation) were fractionated as outlined in (b). Equal protein amounts from each fraction were analyzed by immunobloting using antibodies against the indicated NER proteins. Abundance of H2A is shown as a control for chromatin isolation efficiency. d. UV-tiggered changes in chromatin association of core SFs, identifed by quantitative SILAC-proteomics. Proteomic experiments were performed with HDFs as outlined in (a). The table lists representative examples of SFs that participate in distinct snRNP complexes and their chromatin association in response to UV-irradiation (20 J/m 2 , 1h). U2 ans U5 snRNP-SFs, show signifficantly reduced chromatin association (p≤0.05, Significance B 17 ) and are indicated with a cross. ND=non-detected. e. Abundance of SFs in total cell lysates. Total lysates were prepared from U2Os cells that were mock treated or UV irradiated (20 J/m 2 , 1hr post-irradiation) and SFs abundance was assayed by immunobloting. Abundance of H2A is shown as a loading control. Right: immunoblots. Left: quantification of SF signal intensities normalized to H2A ( n=3 , mean ± S.D., one-way ANOVA / Bonferroni). f. UV-dependent interaction of splicing proteins with elongating RNAPII. Quiescent HDFs were prepared as outlined in (b) exept that, instead of MNase digestion, chromatin was mechanically sheared. Elongating RNAPII was immunoprecipitated with an antibody that recognizes specifically the ser2-phosphorylated RNAPII C-Terminal Domain (CTD) and its interaction with the U2 snRNP-SFs SF3a1 and SF3b2 was assayed by immunoblotting.
    Figure Legend Snippet: a. Schematic overview of the proteomic experiments for the identification of proteins that display UV-dependant chromatin association. b. Schematic outline of cell fractionation. c. Validation of chromatin isolation protocol for NER proteins that are reqruited to chromatin in response to DNA damage. Mock-treated or UV irradiated quiescent HDFs (20 J/m 2 , 1hr post-irradiation) were fractionated as outlined in (b). Equal protein amounts from each fraction were analyzed by immunobloting using antibodies against the indicated NER proteins. Abundance of H2A is shown as a control for chromatin isolation efficiency. d. UV-tiggered changes in chromatin association of core SFs, identifed by quantitative SILAC-proteomics. Proteomic experiments were performed with HDFs as outlined in (a). The table lists representative examples of SFs that participate in distinct snRNP complexes and their chromatin association in response to UV-irradiation (20 J/m 2 , 1h). U2 ans U5 snRNP-SFs, show signifficantly reduced chromatin association (p≤0.05, Significance B 17 ) and are indicated with a cross. ND=non-detected. e. Abundance of SFs in total cell lysates. Total lysates were prepared from U2Os cells that were mock treated or UV irradiated (20 J/m 2 , 1hr post-irradiation) and SFs abundance was assayed by immunobloting. Abundance of H2A is shown as a loading control. Right: immunoblots. Left: quantification of SF signal intensities normalized to H2A ( n=3 , mean ± S.D., one-way ANOVA / Bonferroni). f. UV-dependent interaction of splicing proteins with elongating RNAPII. Quiescent HDFs were prepared as outlined in (b) exept that, instead of MNase digestion, chromatin was mechanically sheared. Elongating RNAPII was immunoprecipitated with an antibody that recognizes specifically the ser2-phosphorylated RNAPII C-Terminal Domain (CTD) and its interaction with the U2 snRNP-SFs SF3a1 and SF3b2 was assayed by immunoblotting.

    Techniques Used: Cell Fractionation, Isolation, Irradiation, Western Blot, Immunoprecipitation

    DNA damage-triggered chromatin-displacement of activated spliceosomes a,b, UV-induced changes in chromatin-association of spliceosome components in quiescent HDFs; a, Immunoblots (right) and quantification (left) of SF chromatin-association; b, chromatin-associated snRNAs quantified by Q-PCR and normalized to HotAir ncRNA ( n=4 , mean ± s.d., T-test). d,e, immunoblots (right) and quantification (left) of SF chromatin-association in U2Os cells; d, time post UV-irradiation, e, UV dose-response and lack of influence of proteasome inhibition. b, d, e , Graphs: Signal intensities normalized to H2A. ( n=3 , mean ± s.d., T-test and one-way ANOVA.
    Figure Legend Snippet: DNA damage-triggered chromatin-displacement of activated spliceosomes a,b, UV-induced changes in chromatin-association of spliceosome components in quiescent HDFs; a, Immunoblots (right) and quantification (left) of SF chromatin-association; b, chromatin-associated snRNAs quantified by Q-PCR and normalized to HotAir ncRNA ( n=4 , mean ± s.d., T-test). d,e, immunoblots (right) and quantification (left) of SF chromatin-association in U2Os cells; d, time post UV-irradiation, e, UV dose-response and lack of influence of proteasome inhibition. b, d, e , Graphs: Signal intensities normalized to H2A. ( n=3 , mean ± s.d., T-test and one-way ANOVA.

    Techniques Used: Western Blot, Polymerase Chain Reaction, Irradiation, Inhibition

    32) Product Images from "Increased global transcription activity as a mechanism of replication stress in cancer"

    Article Title: Increased global transcription activity as a mechanism of replication stress in cancer

    Journal: Nature Communications

    doi: 10.1038/ncomms13087

    TBP is an effector in HRAS V12 -induced replication stress. ( a ) TBP mRNA quantification by quantitative reverse transcriptase–PCR in BJ-HRAS V12 cells 72 h after RAS induction. TBP mRNA levels were normalized to GAPDH and control. ( b ) Protein levels of TBP and β-ACTIN after RAS induction for the times indicated. ( c ) Densitometry quantification of TBP levels based on western blotting as in b after RAS induction for the times indicated. Values were normalized to 72 h control. N =3 (24 and 48 h), N =6 (72 h). ( d ) Twenty-four hours after RAS induction, cells were transfected with TBP siRNA (TBPsi #1) or control siRNA (nonTsi). Cells were processed for DNA fibre analysis or western blotting 48 h later and for 53BP1 staining 24 h later. ( e ) Protein levels of TBP, HRAS and GAPDH (loading control) 72 h after RAS induction and 48 h after siRNA transfection. ( f ) Quantification of nascent RNA synthesis by EU incorporation ±TBPsi #1 72 h after RAS induction. N =3. ( g ) Distribution of replication fork speeds ±TBPsi #1 72 h after RAS induction. N =3. ( h ) Median replication fork speeds ±TBPsi #1 72 h after RAS induction. N =3. ( i ) Percentages of cells containing more than eight 53BP1 foci, ±TBPsi #1 96 h after RAS induction. N =3. ( j ) Median replication fork speeds in cells treated with TBPsi #1 and DRB 72 h after RAS induction, compared with TBPsi #1 or DRB alone. N =3. ( k ) Percentages of cells treated with TBPsi #1 and DRB containing more than eight 53BP1 foci after 96 h after RAS induction, compared with TBPsi #1 or DRB alone. N =3. ( l ) Model for the role of TBP in HRAS V12 -induced replication stress. Means ±s.e.m. (bars) are shown. Student's t -test, * P
    Figure Legend Snippet: TBP is an effector in HRAS V12 -induced replication stress. ( a ) TBP mRNA quantification by quantitative reverse transcriptase–PCR in BJ-HRAS V12 cells 72 h after RAS induction. TBP mRNA levels were normalized to GAPDH and control. ( b ) Protein levels of TBP and β-ACTIN after RAS induction for the times indicated. ( c ) Densitometry quantification of TBP levels based on western blotting as in b after RAS induction for the times indicated. Values were normalized to 72 h control. N =3 (24 and 48 h), N =6 (72 h). ( d ) Twenty-four hours after RAS induction, cells were transfected with TBP siRNA (TBPsi #1) or control siRNA (nonTsi). Cells were processed for DNA fibre analysis or western blotting 48 h later and for 53BP1 staining 24 h later. ( e ) Protein levels of TBP, HRAS and GAPDH (loading control) 72 h after RAS induction and 48 h after siRNA transfection. ( f ) Quantification of nascent RNA synthesis by EU incorporation ±TBPsi #1 72 h after RAS induction. N =3. ( g ) Distribution of replication fork speeds ±TBPsi #1 72 h after RAS induction. N =3. ( h ) Median replication fork speeds ±TBPsi #1 72 h after RAS induction. N =3. ( i ) Percentages of cells containing more than eight 53BP1 foci, ±TBPsi #1 96 h after RAS induction. N =3. ( j ) Median replication fork speeds in cells treated with TBPsi #1 and DRB 72 h after RAS induction, compared with TBPsi #1 or DRB alone. N =3. ( k ) Percentages of cells treated with TBPsi #1 and DRB containing more than eight 53BP1 foci after 96 h after RAS induction, compared with TBPsi #1 or DRB alone. N =3. ( l ) Model for the role of TBP in HRAS V12 -induced replication stress. Means ±s.e.m. (bars) are shown. Student's t -test, * P

    Techniques Used: Polymerase Chain Reaction, Western Blot, Transfection, Staining

    33) Product Images from "RECQ-like helicases Sgs1 and BLM regulate R-loop–associated genome instability"

    Article Title: RECQ-like helicases Sgs1 and BLM regulate R-loop–associated genome instability

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201703168

    R-loop accumulation and DNA damage in BLM-depleted cells. (A and C) Representative images of S9.6 staining in HeLa cells treated with the indicated siRNA targeted for BLM (si-BLM) or a control luciferase (si-Luc; A) or Bloom’s syndrome fibroblasts complemented with an empty vector control (BSF), WT BLM (BSF + WT), or helicase dead mutant (BSF + HM; C). Cells were transfected with either a control vector (GFP) or one expressing GFP-RNaseH1. (B and D) Quantification of S9.6 signal intensity for nuclear area in the indicated conditions in HeLa cells (B) or Bloom’s syndrome fibroblasts (D). Cell numbers scored across three independent replicates are noted below panel B for HeLa cells. (E) RNaseH-dependent DNA breaks in BLM-deficient HeLa cells. (Left) Representative comet tail images from single-cell electrophoresis. (Right) Quantification of comet tail moment under the indicated conditions. t tests were used for comparisons shown. (F and G) Percentages of cells with ≥10 γ-H2AX foci in HeLa cells treated with indicated siRNA expressing GFP or GFP-RNaseH1 (F) or Bloom’s syndrome fibroblasts expressing GFP or GFP-RNaseH1 (G). *, P
    Figure Legend Snippet: R-loop accumulation and DNA damage in BLM-depleted cells. (A and C) Representative images of S9.6 staining in HeLa cells treated with the indicated siRNA targeted for BLM (si-BLM) or a control luciferase (si-Luc; A) or Bloom’s syndrome fibroblasts complemented with an empty vector control (BSF), WT BLM (BSF + WT), or helicase dead mutant (BSF + HM; C). Cells were transfected with either a control vector (GFP) or one expressing GFP-RNaseH1. (B and D) Quantification of S9.6 signal intensity for nuclear area in the indicated conditions in HeLa cells (B) or Bloom’s syndrome fibroblasts (D). Cell numbers scored across three independent replicates are noted below panel B for HeLa cells. (E) RNaseH-dependent DNA breaks in BLM-deficient HeLa cells. (Left) Representative comet tail images from single-cell electrophoresis. (Right) Quantification of comet tail moment under the indicated conditions. t tests were used for comparisons shown. (F and G) Percentages of cells with ≥10 γ-H2AX foci in HeLa cells treated with indicated siRNA expressing GFP or GFP-RNaseH1 (F) or Bloom’s syndrome fibroblasts expressing GFP or GFP-RNaseH1 (G). *, P

    Techniques Used: Staining, Luciferase, Plasmid Preparation, Mutagenesis, Transfection, Expressing, Electrophoresis

    34) Product Images from "Regulation of ATR activity via the RNA polymerase II associated factors CDC73 and PNUTS-PP1"

    Article Title: Regulation of ATR activity via the RNA polymerase II associated factors CDC73 and PNUTS-PP1

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1233

    PNUTS-PP1 suppresses ATR signaling. ( A ) Western blot analysis of ATR and ATM signaling events in control scrambled siRNA transfected (scr) or PNUTS siRNA transfected (siPNUTS #1 and siPNUTS #2) HeLa cells, without IR or at indicated times after 10 Gy. Cells were harvested at 72 h after siRNA transfection. Bottom bar charts show quantification of pCHK1 S317 relative to CHK1 and pRPA S33 relative to γTUBULIN levels for siPNUTS #2, hereafter called siPNUTS ( n = 8). ( B ) Western blot analysis of untreated cells or at 2 or 6 h after addition of thymidine to cells siRNA transfected as in A) (scr and siPNUTS). Bottom bar charts show quantification of pCHK1 S317 relative to CHK1 and pRPA S33 relative to RPA70 levels ( n = 10). ( C ) Western blot analysis of HeLa cells or HeLa BAC clones stably expressing EGFP mouse pnuts (mpnuts) transfected with scr or siPNUTS (specifically targets human PNUTS), without IR or at 1 or 6 h after 10 Gy. Lines to the right of the western blot indicate migration of human endogenous PNUTS (lower band) and EGFP mpnuts (upper band). Bottom bar chart shows quantification of pCHK1 S345 relative to CHK1 levels ( n = 3). ( D ) Western blot analysis of HeLa cells transfected with scr or siPNUTS. At 24 h post transfection, the indicated samples were transfected with wild type EGFP PNUTS or PP1-binding deficient EGFP PNUTS RAXA. Cells were harvested 48 h later without further treatment (–) or 1 h after 10 Gy. Lines to the right of the western blot indicate migration of endogenous PNUTS (lower band) and EGFP PNUTS/EGFP PNUTS RAXA (upper band), asterisk indicates what is likely EGFP PNUTS/EGFP PNUTS RAXA degradation products. Bar chart shows quantification of pCHK1 S317 relative to CHK1 ( n = 3). Error bars indicate standard error of the mean (SEM) and statistical significance was calculated by the two-tailed Student's two sample t-test. * P
    Figure Legend Snippet: PNUTS-PP1 suppresses ATR signaling. ( A ) Western blot analysis of ATR and ATM signaling events in control scrambled siRNA transfected (scr) or PNUTS siRNA transfected (siPNUTS #1 and siPNUTS #2) HeLa cells, without IR or at indicated times after 10 Gy. Cells were harvested at 72 h after siRNA transfection. Bottom bar charts show quantification of pCHK1 S317 relative to CHK1 and pRPA S33 relative to γTUBULIN levels for siPNUTS #2, hereafter called siPNUTS ( n = 8). ( B ) Western blot analysis of untreated cells or at 2 or 6 h after addition of thymidine to cells siRNA transfected as in A) (scr and siPNUTS). Bottom bar charts show quantification of pCHK1 S317 relative to CHK1 and pRPA S33 relative to RPA70 levels ( n = 10). ( C ) Western blot analysis of HeLa cells or HeLa BAC clones stably expressing EGFP mouse pnuts (mpnuts) transfected with scr or siPNUTS (specifically targets human PNUTS), without IR or at 1 or 6 h after 10 Gy. Lines to the right of the western blot indicate migration of human endogenous PNUTS (lower band) and EGFP mpnuts (upper band). Bottom bar chart shows quantification of pCHK1 S345 relative to CHK1 levels ( n = 3). ( D ) Western blot analysis of HeLa cells transfected with scr or siPNUTS. At 24 h post transfection, the indicated samples were transfected with wild type EGFP PNUTS or PP1-binding deficient EGFP PNUTS RAXA. Cells were harvested 48 h later without further treatment (–) or 1 h after 10 Gy. Lines to the right of the western blot indicate migration of endogenous PNUTS (lower band) and EGFP PNUTS/EGFP PNUTS RAXA (upper band), asterisk indicates what is likely EGFP PNUTS/EGFP PNUTS RAXA degradation products. Bar chart shows quantification of pCHK1 S317 relative to CHK1 ( n = 3). Error bars indicate standard error of the mean (SEM) and statistical significance was calculated by the two-tailed Student's two sample t-test. * P

    Techniques Used: Western Blot, Transfection, BAC Assay, Clone Assay, Stable Transfection, Expressing, Migration, Binding Assay, Two Tailed Test

    CDC73, but not TOPBP1 nor ETAA1, is required for high ATR-dependent phosphorylation of both CHK1 and RPA after PNUTS depletion. ( A and B ) Western blot and quantifications ( n = 3) from cells transfected with scr, siPNUTS, and siRNA against TOPBP1 (siTOPBP1) harvested at 72 h after siRNA transfection and 1 and 6 h after 10 Gy. VE-821 was added 30 min prior to 10 Gy. For the siTOPBP1 10 Gy 6 h sample error bar was emitted in the quantifications as experiment was performed two times. Western blot for siTOPBP1 alone is shown in Supplementary Figure S5E . ( C and D ) Western blot and quantifications ( n = 3) from cells transfected with scr, siPNUTS, siTOPBP1 and siRNA against ETAA1 (siETAA1) harvested at 48 h after siRNA transfection and 1 and 6 h after 10 Gy. ( E ) Western blot analysis and quantifications of scr, siPNUTS or CDC73 siRNA (siCDC73) transfected HeLa cells or HeLa cells stably expressing siRNA-resistant Flag-CDC73 treated with IR (10Gy) as indicated. Bar charts show quantification of pCHK1 S345 and pCHK1 S317 versus CHK1 levels at 6 h after 10 Gy ( n = 3). Error bars indicate SEM and statistical significance was calculated by the two-tailed Student's two sample t -test. * P
    Figure Legend Snippet: CDC73, but not TOPBP1 nor ETAA1, is required for high ATR-dependent phosphorylation of both CHK1 and RPA after PNUTS depletion. ( A and B ) Western blot and quantifications ( n = 3) from cells transfected with scr, siPNUTS, and siRNA against TOPBP1 (siTOPBP1) harvested at 72 h after siRNA transfection and 1 and 6 h after 10 Gy. VE-821 was added 30 min prior to 10 Gy. For the siTOPBP1 10 Gy 6 h sample error bar was emitted in the quantifications as experiment was performed two times. Western blot for siTOPBP1 alone is shown in Supplementary Figure S5E . ( C and D ) Western blot and quantifications ( n = 3) from cells transfected with scr, siPNUTS, siTOPBP1 and siRNA against ETAA1 (siETAA1) harvested at 48 h after siRNA transfection and 1 and 6 h after 10 Gy. ( E ) Western blot analysis and quantifications of scr, siPNUTS or CDC73 siRNA (siCDC73) transfected HeLa cells or HeLa cells stably expressing siRNA-resistant Flag-CDC73 treated with IR (10Gy) as indicated. Bar charts show quantification of pCHK1 S345 and pCHK1 S317 versus CHK1 levels at 6 h after 10 Gy ( n = 3). Error bars indicate SEM and statistical significance was calculated by the two-tailed Student's two sample t -test. * P

    Techniques Used: Recombinase Polymerase Amplification, Western Blot, Transfection, Stable Transfection, Expressing, Two Tailed Test

    High ATR signaling after PNUTS depletion is present in individual cells, does not correlate with DNA damage markers and can occur in G1-phase. ( A ) Flow cytometry charts showing γH2AX versus DNA staining of individual scr and siPNUTS transfected cells with and without VE-822 for 1 h. S-phase cells were gated based on DNA content as indicated (black boxes). Quantifications show average median γH2AX levels in S-phase ( n = 3). * P
    Figure Legend Snippet: High ATR signaling after PNUTS depletion is present in individual cells, does not correlate with DNA damage markers and can occur in G1-phase. ( A ) Flow cytometry charts showing γH2AX versus DNA staining of individual scr and siPNUTS transfected cells with and without VE-822 for 1 h. S-phase cells were gated based on DNA content as indicated (black boxes). Quantifications show average median γH2AX levels in S-phase ( n = 3). * P

    Techniques Used: Flow Cytometry, Cytometry, Staining, Transfection

    PNUTS-PP1 likely suppresses ATR signaling by dephosphorylating pRNAPII CTD. ( A ) Western blot analysis of scr or siPNUTS transfected cells without IR or 6 h after 10 Gy. VE-822 was added for 2, 5, 15, 30 or 60 min to indicated samples 6 h after 10 Gy. Charts show fold changes for VE-822-treated samples relative to the 10 Gy 6 h sample, for respective siRNA oligos from quantifications of pCHK1 S317 relative to CHK1 and pRPA S33 relative to CDK1. Experiment was performed 2 times with similar results. ( B ) Western blot analysis of scr and siPNUTS cells at 72 h after transfection. Bottom bar chart shows quantification of pRNAPII S5 relative to RNAPII ( n = 14). *** P
    Figure Legend Snippet: PNUTS-PP1 likely suppresses ATR signaling by dephosphorylating pRNAPII CTD. ( A ) Western blot analysis of scr or siPNUTS transfected cells without IR or 6 h after 10 Gy. VE-822 was added for 2, 5, 15, 30 or 60 min to indicated samples 6 h after 10 Gy. Charts show fold changes for VE-822-treated samples relative to the 10 Gy 6 h sample, for respective siRNA oligos from quantifications of pCHK1 S317 relative to CHK1 and pRPA S33 relative to CDK1. Experiment was performed 2 times with similar results. ( B ) Western blot analysis of scr and siPNUTS cells at 72 h after transfection. Bottom bar chart shows quantification of pRNAPII S5 relative to RNAPII ( n = 14). *** P

    Techniques Used: Western Blot, Transfection

    CDC73 is required for high ATR signaling in S-phase and activation of the endogenous G2 checkpoint after PNUTS depletion, and interacts with ATR and RNAPII. ( A ) Flow cytometry charts showing γH2AX staining versus DNA content as in 3A) of scr, siPNUTS or siPNUTS and siCDC73 transfected cells harvested at 72 h after siRNA transfection with and without 1 h treatment with VE-822. Quantifications show relative median γH2AX levels in indicated S-phase cells (black box). ( n = 3) * P
    Figure Legend Snippet: CDC73 is required for high ATR signaling in S-phase and activation of the endogenous G2 checkpoint after PNUTS depletion, and interacts with ATR and RNAPII. ( A ) Flow cytometry charts showing γH2AX staining versus DNA content as in 3A) of scr, siPNUTS or siPNUTS and siCDC73 transfected cells harvested at 72 h after siRNA transfection with and without 1 h treatment with VE-822. Quantifications show relative median γH2AX levels in indicated S-phase cells (black box). ( n = 3) * P

    Techniques Used: Activation Assay, Flow Cytometry, Cytometry, Staining, Transfection

    35) Product Images from "RNase H1 Regulates Mitochondrial Transcription and Translation via the Degradation of 7S RNA"

    Article Title: RNase H1 Regulates Mitochondrial Transcription and Translation via the Degradation of 7S RNA

    Journal: Frontiers in Genetics

    doi: 10.3389/fgene.2019.01393

    Mitochondrial DNA maintenance in mutant RNASEH1 fibroblasts. (A) Southern blot of total DNA digested with Pvu II from control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium. A radioactive probe against mtDNA was used to detect both linearized mtDNA (empty arrowhead) and 7S DNA (filled arrowhead and bracket), while a probe against 18S rDNA was used as loading control. (B) Relative mitochondrial DNA copy number in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium, calculated as the linearized mtDNA/18S rDNA signal ratio. Data are shown as mean ± SD, n = 4, ***p
    Figure Legend Snippet: Mitochondrial DNA maintenance in mutant RNASEH1 fibroblasts. (A) Southern blot of total DNA digested with Pvu II from control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium. A radioactive probe against mtDNA was used to detect both linearized mtDNA (empty arrowhead) and 7S DNA (filled arrowhead and bracket), while a probe against 18S rDNA was used as loading control. (B) Relative mitochondrial DNA copy number in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium, calculated as the linearized mtDNA/18S rDNA signal ratio. Data are shown as mean ± SD, n = 4, ***p

    Techniques Used: Mutagenesis, Southern Blot

    RNASEH1 mutations, transcript, and protein levels. (A) Domains ofhuman RNAse H1 protein (MTS, mitochondrial targeting sequence; HBD, hybrid binding domain; CD, connection domain; catalytic domain). RNase H1 protein sequences from representative species, H. sapiens (Hs, NP_002927) M. musculus (Mm, NP_035405), B. taurus (Bt, NP_001039970), G. gallus (Gg, NP_990329), X. tropicalis (Xt, NP_001096299), D. rerio (Dr, NP_001002659), C. intestinalis (Ci, F6QPH0), D. melanogaster (Dm, NP_995777), C. elegans (Ce, NP_001040786), S. cerevisiae (Sc, Q04740), were extracted from the database and aligned using ClustalW2. Conserved residues found mutated in the patient in exon 4 are boxed in red, while residues in the active site and interacting with DNA or RNA are boxed in yellow and green, respectively. Positions of β strands are marked by blue arrows. (B) Human RNase H1 crystal structure (PDB ID 2QK9) 18 bp DNA(cyan): RNA (magenta) hybrid is shown respectively. Residues in the active site are colored in yellow. Residues Trp 164 (green) and Val 142 , or the mutated variant Ile 142 (red), are shown as sticks. (C) RNASEH1 transcript levels in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium assessed by qPCR (probe Hs00268000_m1) and normalized to GAPDH transcript levels. Data are shown as mean ± SD, n = 4, ***p
    Figure Legend Snippet: RNASEH1 mutations, transcript, and protein levels. (A) Domains ofhuman RNAse H1 protein (MTS, mitochondrial targeting sequence; HBD, hybrid binding domain; CD, connection domain; catalytic domain). RNase H1 protein sequences from representative species, H. sapiens (Hs, NP_002927) M. musculus (Mm, NP_035405), B. taurus (Bt, NP_001039970), G. gallus (Gg, NP_990329), X. tropicalis (Xt, NP_001096299), D. rerio (Dr, NP_001002659), C. intestinalis (Ci, F6QPH0), D. melanogaster (Dm, NP_995777), C. elegans (Ce, NP_001040786), S. cerevisiae (Sc, Q04740), were extracted from the database and aligned using ClustalW2. Conserved residues found mutated in the patient in exon 4 are boxed in red, while residues in the active site and interacting with DNA or RNA are boxed in yellow and green, respectively. Positions of β strands are marked by blue arrows. (B) Human RNase H1 crystal structure (PDB ID 2QK9) 18 bp DNA(cyan): RNA (magenta) hybrid is shown respectively. Residues in the active site are colored in yellow. Residues Trp 164 (green) and Val 142 , or the mutated variant Ile 142 (red), are shown as sticks. (C) RNASEH1 transcript levels in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium assessed by qPCR (probe Hs00268000_m1) and normalized to GAPDH transcript levels. Data are shown as mean ± SD, n = 4, ***p

    Techniques Used: Sequencing, Binding Assay, Variant Assay, Real-time Polymerase Chain Reaction

    Mitochondrial transcript levels in mutant RNASEH1 fibroblasts. Mitochondrial transcript levels in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium, assessed by qPCR and normalized to GAPDH transcript levels. Analyzed transcripts included the non-coding 7S RNA (MT-7S), the two ribosomal RNAs MT-RNR1 (12S rRNA) and MT-RNR2 (16S rRNA), three complex IV protein mRNAs (MT-CO1, MT-CO2 and MT-CO3), three complex I protein mRNAs (MT-ND1, MT-ND5 and MT-ND6), one complex III protein mRNA (MT-CYB), and one complex V protein mRNA (MT-ATP6). Data are shown as mean ± SD, n = 4, Student’s unpaired two-tail t-test, **p
    Figure Legend Snippet: Mitochondrial transcript levels in mutant RNASEH1 fibroblasts. Mitochondrial transcript levels in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium, assessed by qPCR and normalized to GAPDH transcript levels. Analyzed transcripts included the non-coding 7S RNA (MT-7S), the two ribosomal RNAs MT-RNR1 (12S rRNA) and MT-RNR2 (16S rRNA), three complex IV protein mRNAs (MT-CO1, MT-CO2 and MT-CO3), three complex I protein mRNAs (MT-ND1, MT-ND5 and MT-ND6), one complex III protein mRNA (MT-CYB), and one complex V protein mRNA (MT-ATP6). Data are shown as mean ± SD, n = 4, Student’s unpaired two-tail t-test, **p

    Techniques Used: Mutagenesis, Real-time Polymerase Chain Reaction

    Mitochondrial translation in mutant RNASEH1 fibroblasts. (A) Western blot analysis of mitochondrial proteins involved in mitochondrial RNA metabolism (RNA metab.) and mitochondrial large (mtLSU) and small (mtSSU) ribosomal subunits in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium. GAPDH was used as loading control. GAPDH is from the same blot as Figure1D . (B) Quantification of the Western blots shown in (A) normalized to GAPDH levels. Data are shown as mean ± SD, Student’s unpaired two-tail t-test, *p
    Figure Legend Snippet: Mitochondrial translation in mutant RNASEH1 fibroblasts. (A) Western blot analysis of mitochondrial proteins involved in mitochondrial RNA metabolism (RNA metab.) and mitochondrial large (mtLSU) and small (mtSSU) ribosomal subunits in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium. GAPDH was used as loading control. GAPDH is from the same blot as Figure1D . (B) Quantification of the Western blots shown in (A) normalized to GAPDH levels. Data are shown as mean ± SD, Student’s unpaired two-tail t-test, *p

    Techniques Used: Mutagenesis, Western Blot

    Mitochondrial DNA maintenance in mutant RNASEH1 fibroblasts. (A) Western blot analysis of representative components of the mitochondrial OxPhos complexes I-V in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium. GAPDH was used as loading control. (B) Oxygen consumption ( I O2 ) measurements in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium. Values of basal and maximal respiration along with ATP production-dependent, proton leak respiration, and spare capacity are presented. Data are shown as mean ± SD, n = 4, Student’s unpaired two-tail t-test, **p
    Figure Legend Snippet: Mitochondrial DNA maintenance in mutant RNASEH1 fibroblasts. (A) Western blot analysis of representative components of the mitochondrial OxPhos complexes I-V in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium. GAPDH was used as loading control. (B) Oxygen consumption ( I O2 ) measurements in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium. Values of basal and maximal respiration along with ATP production-dependent, proton leak respiration, and spare capacity are presented. Data are shown as mean ± SD, n = 4, Student’s unpaired two-tail t-test, **p

    Techniques Used: Mutagenesis, Western Blot

    36) Product Images from "The Hepatitis B Virus Ribonuclease H Is Sensitive to Inhibitors of the Human Immunodeficiency Virus Ribonuclease H and Integrase Enzymes"

    Article Title: The Hepatitis B Virus Ribonuclease H Is Sensitive to Inhibitors of the Human Immunodeficiency Virus Ribonuclease H and Integrase Enzymes

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003125

    Activity of HBV RNAseH inhibitors against human RNAseH1. A. Proteins in the enriched recombinant human RNAseH1 lysates employed in the RNAseH reactions were detected by Coomassie-blue staining following SDS-PAGE. B. An oligonucleotide-directed RNAseH assay was conducted with wild-type HBV RNAseH (genotype D) and recombinant human RNAseH1 under identical reaction conditions. The inhibitory compounds were employed at 10 µM. The upper and lower panels are from the same experiment and the data were collected on a single sheet of film, so the reactions can be directly compared. DMSO, vehicle control. S, the DRF+ substrate; P1 and P2, RNAseH cleavage products.
    Figure Legend Snippet: Activity of HBV RNAseH inhibitors against human RNAseH1. A. Proteins in the enriched recombinant human RNAseH1 lysates employed in the RNAseH reactions were detected by Coomassie-blue staining following SDS-PAGE. B. An oligonucleotide-directed RNAseH assay was conducted with wild-type HBV RNAseH (genotype D) and recombinant human RNAseH1 under identical reaction conditions. The inhibitory compounds were employed at 10 µM. The upper and lower panels are from the same experiment and the data were collected on a single sheet of film, so the reactions can be directly compared. DMSO, vehicle control. S, the DRF+ substrate; P1 and P2, RNAseH cleavage products.

    Techniques Used: Activity Assay, Recombinant, Staining, SDS Page

    Specificity of anti-HBV RNAseH compounds. A. Inhibition of HBV genotype D RNAseH by irrelevant compounds at 10 µM in the oligonucleotide-directed RNAseH assay. Compound #4 was employed as an example HBV RNAseH inhibitor. B. Anti-HBV RNAseH inhibitors do not significantly inhibit the HCV RNA polymerase. The ability of compounds #5, 6 and 8 to inhibit production of poly-G by the HCV RNA-directed RNA polymerase was measured in a primed homopolymeric RNA synthesis assay [82] . The compounds were employed at 10 µM. DMSO, vehicle control. C. Dose-responsiveness of HBV RNAseH inhibition. The effects of compounds #6, 8, and 12 on the RNAseH activity of HRHPL (genotype D) were measured at concentrations ranging from 0.5 to 50 µM. The dose-response profile is plotted for compound #12.
    Figure Legend Snippet: Specificity of anti-HBV RNAseH compounds. A. Inhibition of HBV genotype D RNAseH by irrelevant compounds at 10 µM in the oligonucleotide-directed RNAseH assay. Compound #4 was employed as an example HBV RNAseH inhibitor. B. Anti-HBV RNAseH inhibitors do not significantly inhibit the HCV RNA polymerase. The ability of compounds #5, 6 and 8 to inhibit production of poly-G by the HCV RNA-directed RNA polymerase was measured in a primed homopolymeric RNA synthesis assay [82] . The compounds were employed at 10 µM. DMSO, vehicle control. C. Dose-responsiveness of HBV RNAseH inhibition. The effects of compounds #6, 8, and 12 on the RNAseH activity of HRHPL (genotype D) were measured at concentrations ranging from 0.5 to 50 µM. The dose-response profile is plotted for compound #12.

    Techniques Used: Inhibition, Activity Assay

    Recombinant RNAseHs from HBV genotypes A, B, C, D, and H. A. Sequence alignment for genotype A, B, C, D, and H versions of the HBV RNAseH expression construct HRHPL. The additional methionine at residue 10 of the genotype D sequence is a product of the cloning strategy; this insertion has no impact on the RNAseH activity because the first 9 amino acids of HRHPL can be deleted without altering the biochemical profile of the enzyme. * indicates the DEDD active site residues, and the hexahistidine tag at the C-terminus is underlined. Residue 1 for the HBV RNAseH domain is amino acid 684 in the full-length polymerase protein (strain adw2). B. Western analysis of RNAseH proteins in the enriched lysates probed with the anti-HBV RNAseH monoclonal antibody 9F9. C. RNAseH activity of RNAseH from HBV genotypes A, B, C, D, and H detected by the oligonucleotide-directed RNA cleavage assay. HRHPL-D702A (genotype D) is a negative control. gt, genotype.
    Figure Legend Snippet: Recombinant RNAseHs from HBV genotypes A, B, C, D, and H. A. Sequence alignment for genotype A, B, C, D, and H versions of the HBV RNAseH expression construct HRHPL. The additional methionine at residue 10 of the genotype D sequence is a product of the cloning strategy; this insertion has no impact on the RNAseH activity because the first 9 amino acids of HRHPL can be deleted without altering the biochemical profile of the enzyme. * indicates the DEDD active site residues, and the hexahistidine tag at the C-terminus is underlined. Residue 1 for the HBV RNAseH domain is amino acid 684 in the full-length polymerase protein (strain adw2). B. Western analysis of RNAseH proteins in the enriched lysates probed with the anti-HBV RNAseH monoclonal antibody 9F9. C. RNAseH activity of RNAseH from HBV genotypes A, B, C, D, and H detected by the oligonucleotide-directed RNA cleavage assay. HRHPL-D702A (genotype D) is a negative control. gt, genotype.

    Techniques Used: Recombinant, Sequencing, Expressing, Construct, Clone Assay, Activity Assay, Western Blot, Cleavage Assay, Negative Control

    Recombinant HBV RNAseH is enzymatically active. A. Oligonucleotide-directed RNAseH assay. Uniformly 32 P-labeled RNA (blue or red) is annealed to a complementary DNA oligonucleotide (black). RNAseH activity cleaves the RNA in the heteroduplex formed where the oligonucleotide anneals to the RNA and yields two products (P1 and P2). B. Recombinant HBV RNAseH is active. An oligonucleotide-directed RNAseH assay was conducted with E. coli RNAseH, wild-type HBV RNAseH (HRHPL), or RNAseH-deficient HRHPL (D702A). A complementary oligonucleotide (+) or non-complementary oligonucleotide (−) was mixed with labeled DRF+ RNA and the reactions were incubated to allow RNAseH activity. The products were resolved by SDS-PAGE and the RNAs were detected by autoradiography. Oligonucleotide set 1 was D2507− and D2526+ and oligonucleotide set #2 was D2543M-Sal and D2453+. The positions of the cleavage products (P1 and P2) are indicated in blue for reactions containing oligonucleotide D2507− and in red for reactions containing oligonucleotide D2543M-Sal. C. FRET-based RNAseH assay. A self-complementary chimeric RNA:DNA synthetic oligonucleotide (RHF1) forms a stem-loop in which the stem is an RNA:DNA heteroduplex. The stem brings the fluorescein (F) and quencher (Q) at the 5′ and 3′ ends of the oligonucleotide into close proximity. Cleavage of the RNA releases the fluorescein and increases its fluorescence. D. Detection of HBV RNAseH activity employing the fluorescent assay. The substrate in panel C was employed in an RNAseH assay employing buffer alone, wild-type HBV RNAseH (HRHPL), or RNAseH-deficient HRHPL (D702A/E731A). *, P
    Figure Legend Snippet: Recombinant HBV RNAseH is enzymatically active. A. Oligonucleotide-directed RNAseH assay. Uniformly 32 P-labeled RNA (blue or red) is annealed to a complementary DNA oligonucleotide (black). RNAseH activity cleaves the RNA in the heteroduplex formed where the oligonucleotide anneals to the RNA and yields two products (P1 and P2). B. Recombinant HBV RNAseH is active. An oligonucleotide-directed RNAseH assay was conducted with E. coli RNAseH, wild-type HBV RNAseH (HRHPL), or RNAseH-deficient HRHPL (D702A). A complementary oligonucleotide (+) or non-complementary oligonucleotide (−) was mixed with labeled DRF+ RNA and the reactions were incubated to allow RNAseH activity. The products were resolved by SDS-PAGE and the RNAs were detected by autoradiography. Oligonucleotide set 1 was D2507− and D2526+ and oligonucleotide set #2 was D2543M-Sal and D2453+. The positions of the cleavage products (P1 and P2) are indicated in blue for reactions containing oligonucleotide D2507− and in red for reactions containing oligonucleotide D2543M-Sal. C. FRET-based RNAseH assay. A self-complementary chimeric RNA:DNA synthetic oligonucleotide (RHF1) forms a stem-loop in which the stem is an RNA:DNA heteroduplex. The stem brings the fluorescein (F) and quencher (Q) at the 5′ and 3′ ends of the oligonucleotide into close proximity. Cleavage of the RNA releases the fluorescein and increases its fluorescence. D. Detection of HBV RNAseH activity employing the fluorescent assay. The substrate in panel C was employed in an RNAseH assay employing buffer alone, wild-type HBV RNAseH (HRHPL), or RNAseH-deficient HRHPL (D702A/E731A). *, P

    Techniques Used: Recombinant, Labeling, Activity Assay, Incubation, SDS Page, Autoradiography, Fluorescence

    37) Product Images from "Two familial ALS proteins function in prevention/repair of transcription-associated DNA damage"

    Article Title: Two familial ALS proteins function in prevention/repair of transcription-associated DNA damage

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1611673113

    FUS localizes to sites of post-UV transcription-associated DNA damage. ( A and B ) U2OS cells were treated with 0 J or 25 J, allowed to recover at 37 °C for 4 h, fixed with methanol:acetic acid, and costained for FUS and RNA Polymerase II phospho-S5
    Figure Legend Snippet: FUS localizes to sites of post-UV transcription-associated DNA damage. ( A and B ) U2OS cells were treated with 0 J or 25 J, allowed to recover at 37 °C for 4 h, fixed with methanol:acetic acid, and costained for FUS and RNA Polymerase II phospho-S5

    Techniques Used:

    TDP43 localizes to sites of post-UV transcription associated DNA damage and colocalizes with FUS. ( A and B ) U2OS cells were exposed to 0 J or 25 J, allowed to recover at 37 °C for 4 h, fixed with methanol:acetic acid, and costained for TDP43 and
    Figure Legend Snippet: TDP43 localizes to sites of post-UV transcription associated DNA damage and colocalizes with FUS. ( A and B ) U2OS cells were exposed to 0 J or 25 J, allowed to recover at 37 °C for 4 h, fixed with methanol:acetic acid, and costained for TDP43 and

    Techniques Used:

    Depletion of fALS proteins leads to increased sensitivity to a transcription stalling agent. ( A ) Western blot demonstrating that FUS siRNAs deplete FUS protein. ( Top ) Blot shows FUS protein levels in U2OS cells transfected with a control siRNA and the
    Figure Legend Snippet: Depletion of fALS proteins leads to increased sensitivity to a transcription stalling agent. ( A ) Western blot demonstrating that FUS siRNAs deplete FUS protein. ( Top ) Blot shows FUS protein levels in U2OS cells transfected with a control siRNA and the

    Techniques Used: Western Blot, Transfection

    FUS and TDP43 are involved in the prevention or repair of R loop-associated DNA damage. ( A ) Representative images of U2OS cells cotransfected with siGL2 siRNA and either an empty vector (vec) or an RNASEH1-encoding plasmid (RNH) and stained for RNASEH1.
    Figure Legend Snippet: FUS and TDP43 are involved in the prevention or repair of R loop-associated DNA damage. ( A ) Representative images of U2OS cells cotransfected with siGL2 siRNA and either an empty vector (vec) or an RNASEH1-encoding plasmid (RNH) and stained for RNASEH1.

    Techniques Used: Plasmid Preparation, Staining

    FUS localizes to sites of post-UV transcription-associated DNA damage with BRCA1. ( A and B ) U2OS cells were exposed to 0 J or 25 J, allowed to recover at 37 °C for 4 h, fixed with methanol:acetic acid, and costained for FUS and phosphorylated
    Figure Legend Snippet: FUS localizes to sites of post-UV transcription-associated DNA damage with BRCA1. ( A and B ) U2OS cells were exposed to 0 J or 25 J, allowed to recover at 37 °C for 4 h, fixed with methanol:acetic acid, and costained for FUS and phosphorylated

    Techniques Used:

    Antibody used for detection of TDP43. ( A–C ) U2OS cells were plated on coverslips on day 1, transfected with either a control siRNA (siGL2-A) or one of two different TDP43-specific siRNAs (siTDP43 #2-B or siTDP43 #3-C) on days 2 and 3, and fixed
    Figure Legend Snippet: Antibody used for detection of TDP43. ( A–C ) U2OS cells were plated on coverslips on day 1, transfected with either a control siRNA (siGL2-A) or one of two different TDP43-specific siRNAs (siTDP43 #2-B or siTDP43 #3-C) on days 2 and 3, and fixed

    Techniques Used: Transfection

    TDP43 localizes to sites of post-UV transcription-associated DNA damage. ( A and B ) U2OS cells were exposed to 0 J or 25 J, allowed to recover at 37 °C for 4 h, fixed with methanol:acetic acid, and costained for TDP43 and RNA Polymerase II phospho-S5
    Figure Legend Snippet: TDP43 localizes to sites of post-UV transcription-associated DNA damage. ( A and B ) U2OS cells were exposed to 0 J or 25 J, allowed to recover at 37 °C for 4 h, fixed with methanol:acetic acid, and costained for TDP43 and RNA Polymerase II phospho-S5

    Techniques Used:

    38) Product Images from "Purification and enzymatic characterization of the hepatitis B virus ribonuclease H, a new target for antiviral inhibitors"

    Article Title: Purification and enzymatic characterization of the hepatitis B virus ribonuclease H, a new target for antiviral inhibitors

    Journal: Antiviral research

    doi: 10.1016/j.antiviral.2016.06.005

    Minimal heteroduplex size recognized by the HBV RNaseH ODN-directed cleavage assays were performed with ODNs of different lengths (20 nt to 7 nt) for: A , HBV MBP-HRHgtC; B , HBV MBP-HRHgtCΔ5; C , Human RNaseH1; D , E. coli RNaseH1. −, non-complementary control ODN; +, complementary ODN; S, substrate; P1, product 1; P2, product 2.
    Figure Legend Snippet: Minimal heteroduplex size recognized by the HBV RNaseH ODN-directed cleavage assays were performed with ODNs of different lengths (20 nt to 7 nt) for: A , HBV MBP-HRHgtC; B , HBV MBP-HRHgtCΔ5; C , Human RNaseH1; D , E. coli RNaseH1. −, non-complementary control ODN; +, complementary ODN; S, substrate; P1, product 1; P2, product 2.

    Techniques Used:

    39) Product Images from "Purification and enzymatic characterization of the hepatitis B virus ribonuclease H, a new target for antiviral inhibitors"

    Article Title: Purification and enzymatic characterization of the hepatitis B virus ribonuclease H, a new target for antiviral inhibitors

    Journal: Antiviral research

    doi: 10.1016/j.antiviral.2016.06.005

    Minimal heteroduplex size recognized by the HBV RNaseH ODN-directed cleavage assays were performed with ODNs of different lengths (20 nt to 7 nt) for: A , HBV MBP-HRHgtC; B , HBV MBP-HRHgtCΔ5; C , Human RNaseH1; D , E. coli RNaseH1. −, non-complementary control ODN; +, complementary ODN; S, substrate; P1, product 1; P2, product 2.
    Figure Legend Snippet: Minimal heteroduplex size recognized by the HBV RNaseH ODN-directed cleavage assays were performed with ODNs of different lengths (20 nt to 7 nt) for: A , HBV MBP-HRHgtC; B , HBV MBP-HRHgtCΔ5; C , Human RNaseH1; D , E. coli RNaseH1. −, non-complementary control ODN; +, complementary ODN; S, substrate; P1, product 1; P2, product 2.

    Techniques Used:

    40) Product Images from "Drosha drives the formation of DNA:RNA hybrids around DNA break sites to facilitate DNA repair"

    Article Title: Drosha drives the formation of DNA:RNA hybrids around DNA break sites to facilitate DNA repair

    Journal: Nature Communications

    doi: 10.1038/s41467-018-02893-x

    DNA:RNA hybrids form around DNA break sites to facilitate DNA repair in a Drosha-dependent manner. a Relocation of inactivated E. coli mCherry-RNase H1 D10R E48R to sites of laser-induced DNA damage. Representative fluorescence images, top. Scale bars, 10 µm. Bottom, graph showing quantitation of 168 cells over 3 replicates, error bars = SEM. b DNA:RNA hybrid IP (DRIP) followed by qPCR around HR and NHEJ DNA break sites, and control undamaged actin exon 5 locus after 2 h of damage induction. As a positive control, samples were treated in vitro with RNase H1 (shaded section). Error bars = SEM, Student’s paired T -test, * p ≤ 0.05 in 4 biological replicates. c DRIP-Seq was performed in conditions as in b . Graph shows enrichment of DNA:RNA hybrids around HR-repaired and NHEJ-repaired cut sites following DNA damage compared to sites documented to remain uncut following damage induction. d Over-expression of RNase H1 or a GFP control was followed by DNA resection assay as in Fig.5 a, b. N = 3, error bars = SEM, Student’s 2-sample T -test, ** p ≤ 0.01. e RNase H1 was over-expressed in the HR (left) and NHEJ (right) repair reporter system cell lines (described in Fig. 4a ) 6 h prior to I-SceI expression and GFP-positive cells were quantified as a measure of repair efficiency. N = 3 each, error bars = SD, Student’s 2-sample T -test, * p ≤ 0.05
    Figure Legend Snippet: DNA:RNA hybrids form around DNA break sites to facilitate DNA repair in a Drosha-dependent manner. a Relocation of inactivated E. coli mCherry-RNase H1 D10R E48R to sites of laser-induced DNA damage. Representative fluorescence images, top. Scale bars, 10 µm. Bottom, graph showing quantitation of 168 cells over 3 replicates, error bars = SEM. b DNA:RNA hybrid IP (DRIP) followed by qPCR around HR and NHEJ DNA break sites, and control undamaged actin exon 5 locus after 2 h of damage induction. As a positive control, samples were treated in vitro with RNase H1 (shaded section). Error bars = SEM, Student’s paired T -test, * p ≤ 0.05 in 4 biological replicates. c DRIP-Seq was performed in conditions as in b . Graph shows enrichment of DNA:RNA hybrids around HR-repaired and NHEJ-repaired cut sites following DNA damage compared to sites documented to remain uncut following damage induction. d Over-expression of RNase H1 or a GFP control was followed by DNA resection assay as in Fig.5 a, b. N = 3, error bars = SEM, Student’s 2-sample T -test, ** p ≤ 0.01. e RNase H1 was over-expressed in the HR (left) and NHEJ (right) repair reporter system cell lines (described in Fig. 4a ) 6 h prior to I-SceI expression and GFP-positive cells were quantified as a measure of repair efficiency. N = 3 each, error bars = SD, Student’s 2-sample T -test, * p ≤ 0.05

    Techniques Used: Fluorescence, Quantitation Assay, Real-time Polymerase Chain Reaction, Non-Homologous End Joining, Positive Control, In Vitro, Over Expression, Resection Assay, Expressing

    Related Articles

    Clone Assay:

    Article Title: Fusion to Flaviviral Leader Peptide Targets HIV-1 Reverse Transcriptase for Secretion and Reduces Its Enzymatic Activity and Ability to Induce Oxidative Stress but Has No Major Effects on Its Immunogenic Performance in DNA-Immunized Mice
    Article Snippet: .. Plasmids Expression-optimized gene encoding reverse transcriptase derived from the patient infected with multidrug-resistant HIV-1 clade B isolate (RT1.14, [ ]) with mutations D185N, D186N, and E478Q abrogating polymerase and RNase H activities was cloned into pVax1 vector (Invitrogen, USA) generating pVaxRT1.14opt-in which was described by us earlier [ ]. .. Sequence encoding RT1.14opt-in was used as a backbone to design a chimeric gene encoding RT1.14 with the N-terminal insertion of the leader sequence of NS1 protein of TBE.

    Transfection:

    Article Title: Hsp90 protein interacts with phosphorothioate oligonucleotides containing hydrophobic 2′-modifications and enhances antisense activity
    Article Snippet: .. Eight or 24 h after transfection, cells were reseeded at ∼60% confluency in 24-well plates and incubated for 16 h. Next, RNase H1-dependent gapmer ASOs were transfected into cells using Lipofectamine 2000 (Life Technologies), at different final concentrations as indicated in Figures and . .. Four hours after ASO transfection, cells were harvested and RNA or protein was prepared for subsequent analyses.

    Infection:

    Article Title: Fusion to Flaviviral Leader Peptide Targets HIV-1 Reverse Transcriptase for Secretion and Reduces Its Enzymatic Activity and Ability to Induce Oxidative Stress but Has No Major Effects on Its Immunogenic Performance in DNA-Immunized Mice
    Article Snippet: .. Plasmids Expression-optimized gene encoding reverse transcriptase derived from the patient infected with multidrug-resistant HIV-1 clade B isolate (RT1.14, [ ]) with mutations D185N, D186N, and E478Q abrogating polymerase and RNase H activities was cloned into pVax1 vector (Invitrogen, USA) generating pVaxRT1.14opt-in which was described by us earlier [ ]. .. Sequence encoding RT1.14opt-in was used as a backbone to design a chimeric gene encoding RT1.14 with the N-terminal insertion of the leader sequence of NS1 protein of TBE.

    Plasmid Preparation:

    Article Title: Fusion to Flaviviral Leader Peptide Targets HIV-1 Reverse Transcriptase for Secretion and Reduces Its Enzymatic Activity and Ability to Induce Oxidative Stress but Has No Major Effects on Its Immunogenic Performance in DNA-Immunized Mice
    Article Snippet: .. Plasmids Expression-optimized gene encoding reverse transcriptase derived from the patient infected with multidrug-resistant HIV-1 clade B isolate (RT1.14, [ ]) with mutations D185N, D186N, and E478Q abrogating polymerase and RNase H activities was cloned into pVax1 vector (Invitrogen, USA) generating pVaxRT1.14opt-in which was described by us earlier [ ]. .. Sequence encoding RT1.14opt-in was used as a backbone to design a chimeric gene encoding RT1.14 with the N-terminal insertion of the leader sequence of NS1 protein of TBE.

    Incubation:

    Article Title: Hsp90 protein interacts with phosphorothioate oligonucleotides containing hydrophobic 2′-modifications and enhances antisense activity
    Article Snippet: .. Eight or 24 h after transfection, cells were reseeded at ∼60% confluency in 24-well plates and incubated for 16 h. Next, RNase H1-dependent gapmer ASOs were transfected into cells using Lipofectamine 2000 (Life Technologies), at different final concentrations as indicated in Figures and . .. Four hours after ASO transfection, cells were harvested and RNA or protein was prepared for subsequent analyses.

    Activity Assay:

    Article Title: Angiogenin/ribonuclease 5 is an EGFR ligand and a serum biomarker for erlotinib sensitivity in pancreatic cancer
    Article Snippet: .. RNase activity was detected with use of Ambion® RNaseAlert® Lab Test kit (Invitrogen) with a slight modification. ..

    Construct:

    Article Title: RNase H sequence preferences influence antisense oligonucleotide efficiency
    Article Snippet: .. Nevertheless, only one of the modified nucleotides directly interacts with the RNase H1 enzyme ( ) ( ) and reassuringly, for the human and E. coli RNase H, we obtained similar results with all three constructs having different modification patterns (Figure and ), suggesting that the different modifications in the substrates did not distort the results. .. Human RNase H1 has two nucleic acid binding domains: the catalytic domain, which cleaves RNA and is structurally similar to the E. coli RNase H1 ( , , ) and the HBD, which allows for the enzyme’s processive action ( ).

    Expressing:

    Article Title: Fusion to Flaviviral Leader Peptide Targets HIV-1 Reverse Transcriptase for Secretion and Reduces Its Enzymatic Activity and Ability to Induce Oxidative Stress but Has No Major Effects on Its Immunogenic Performance in DNA-Immunized Mice
    Article Snippet: .. Plasmids Expression-optimized gene encoding reverse transcriptase derived from the patient infected with multidrug-resistant HIV-1 clade B isolate (RT1.14, [ ]) with mutations D185N, D186N, and E478Q abrogating polymerase and RNase H activities was cloned into pVax1 vector (Invitrogen, USA) generating pVaxRT1.14opt-in which was described by us earlier [ ]. .. Sequence encoding RT1.14opt-in was used as a backbone to design a chimeric gene encoding RT1.14 with the N-terminal insertion of the leader sequence of NS1 protein of TBE.

    Modification:

    Article Title: RNase H sequence preferences influence antisense oligonucleotide efficiency
    Article Snippet: .. Nevertheless, only one of the modified nucleotides directly interacts with the RNase H1 enzyme ( ) ( ) and reassuringly, for the human and E. coli RNase H, we obtained similar results with all three constructs having different modification patterns (Figure and ), suggesting that the different modifications in the substrates did not distort the results. .. Human RNase H1 has two nucleic acid binding domains: the catalytic domain, which cleaves RNA and is structurally similar to the E. coli RNase H1 ( , , ) and the HBD, which allows for the enzyme’s processive action ( ).

    Article Title: Angiogenin/ribonuclease 5 is an EGFR ligand and a serum biomarker for erlotinib sensitivity in pancreatic cancer
    Article Snippet: .. RNase activity was detected with use of Ambion® RNaseAlert® Lab Test kit (Invitrogen) with a slight modification. ..

    Derivative Assay:

    Article Title: Fusion to Flaviviral Leader Peptide Targets HIV-1 Reverse Transcriptase for Secretion and Reduces Its Enzymatic Activity and Ability to Induce Oxidative Stress but Has No Major Effects on Its Immunogenic Performance in DNA-Immunized Mice
    Article Snippet: .. Plasmids Expression-optimized gene encoding reverse transcriptase derived from the patient infected with multidrug-resistant HIV-1 clade B isolate (RT1.14, [ ]) with mutations D185N, D186N, and E478Q abrogating polymerase and RNase H activities was cloned into pVax1 vector (Invitrogen, USA) generating pVaxRT1.14opt-in which was described by us earlier [ ]. .. Sequence encoding RT1.14opt-in was used as a backbone to design a chimeric gene encoding RT1.14 with the N-terminal insertion of the leader sequence of NS1 protein of TBE.

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  • 85
    Thermo Fisher gene exp rnaseh1 hs00268000 m1
    Mitochondrial DNA maintenance in mutant <t>RNASEH1</t> fibroblasts. (A) Southern blot of total DNA digested with Pvu II from control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium. A radioactive probe against mtDNA was used to detect both linearized mtDNA (empty arrowhead) and 7S DNA (filled arrowhead and bracket), while a probe against 18S rDNA was used as loading control. (B) Relative mitochondrial DNA copy number in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium, calculated as the linearized mtDNA/18S rDNA signal ratio. Data are shown as mean ± SD, n = 4, ***p
    Gene Exp Rnaseh1 Hs00268000 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Thermo Fisher rnase h 5 u µl
    m 5 C mRNA methylation is enriched at transcriptionally active sites with DNA damage. a U2OS-TRE cells transfected with TA-KR/TA-Cherry/tetR-KR/tetR-Cherry plasmids were exposed to light for 30 min for KR activation and allowed to recover for 1 h before harvest (scale bar: 10 μm). Quantification of frequency of cells in 500 cells with m 5 C foci from three independent experiments, mean ± SD (upper right). Fold increase of m 5 C mean intensity = mean intensity of m 5 C at TA-KR/mean intensity of background ( n = 20, mean ± SD) (lower right). b U2OS-TRE cells were transfected with TA-KR/TA-Cherry to induce local oxidative damage or for the control condition. Cells were then stained for m 5 C with four different anti-m 5 C antibodies. Frequency of m 5 C-positive cells in 500 cells was quantified ( n = 3, mean ± SD). c U2OS-TRE cells transfected with TA-KR were digested with <t>RNaseH1,</t> RNaseA, or DNase I and stained for m 5 C quantification (scale bar: 10 μm). d The mRNA from Flp-in 293 cells treated with or without 2 mM H 2 O 2 for 40 min was used for m 5 C measurement via dot blot. Quantification of m 5 C levels (mean ± SD) from three independent experiments normalized with Ctrl and methylene blue is shown. e 32 P-labeled mRNA monophosphate nucleosides were run on 2D gels for 2D-TLC analysis. In vitro-transcribed 4B mRNA with or without m 5 C was run in parallel. Representative images from three sets of independent experiments are shown with arrows showing the directions of each solvent run. Position of each nucleotide and m 5 C are labeled (Left). f 32 P-labeled mRNA monophosphate nucleosides from U2OS cells with or without 2 mM H 2 O 2 for 40 min were run on 2D gels for 2D-TLC analysis. Representative images from three sets of independent experiments. Associated quantification of relative increase in m 5 C in peroxide-treated cells compared to control, normalized to nucleotide C (right). Statistical analysis was performed with the unpaired two tailed Student’s t -test. * p
    Rnase H 5 U µl, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Mitochondrial DNA maintenance in mutant RNASEH1 fibroblasts. (A) Southern blot of total DNA digested with Pvu II from control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium. A radioactive probe against mtDNA was used to detect both linearized mtDNA (empty arrowhead) and 7S DNA (filled arrowhead and bracket), while a probe against 18S rDNA was used as loading control. (B) Relative mitochondrial DNA copy number in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium, calculated as the linearized mtDNA/18S rDNA signal ratio. Data are shown as mean ± SD, n = 4, ***p

    Journal: Frontiers in Genetics

    Article Title: RNase H1 Regulates Mitochondrial Transcription and Translation via the Degradation of 7S RNA

    doi: 10.3389/fgene.2019.01393

    Figure Lengend Snippet: Mitochondrial DNA maintenance in mutant RNASEH1 fibroblasts. (A) Southern blot of total DNA digested with Pvu II from control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium. A radioactive probe against mtDNA was used to detect both linearized mtDNA (empty arrowhead) and 7S DNA (filled arrowhead and bracket), while a probe against 18S rDNA was used as loading control. (B) Relative mitochondrial DNA copy number in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium, calculated as the linearized mtDNA/18S rDNA signal ratio. Data are shown as mean ± SD, n = 4, ***p

    Article Snippet: In order to investigate if the presence of a nonsense mutation was triggering nonsense-mediated decay, we checked RNASEH1 transcript levels in human control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium with probe Hs00268000_m1, spanning exons 7-8 ( ).

    Techniques: Mutagenesis, Southern Blot

    RNASEH1 mutations, transcript, and protein levels. (A) Domains ofhuman RNAse H1 protein (MTS, mitochondrial targeting sequence; HBD, hybrid binding domain; CD, connection domain; catalytic domain). RNase H1 protein sequences from representative species, H. sapiens (Hs, NP_002927) M. musculus (Mm, NP_035405), B. taurus (Bt, NP_001039970), G. gallus (Gg, NP_990329), X. tropicalis (Xt, NP_001096299), D. rerio (Dr, NP_001002659), C. intestinalis (Ci, F6QPH0), D. melanogaster (Dm, NP_995777), C. elegans (Ce, NP_001040786), S. cerevisiae (Sc, Q04740), were extracted from the database and aligned using ClustalW2. Conserved residues found mutated in the patient in exon 4 are boxed in red, while residues in the active site and interacting with DNA or RNA are boxed in yellow and green, respectively. Positions of β strands are marked by blue arrows. (B) Human RNase H1 crystal structure (PDB ID 2QK9) 18 bp DNA(cyan): RNA (magenta) hybrid is shown respectively. Residues in the active site are colored in yellow. Residues Trp 164 (green) and Val 142 , or the mutated variant Ile 142 (red), are shown as sticks. (C) RNASEH1 transcript levels in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium assessed by qPCR (probe Hs00268000_m1) and normalized to GAPDH transcript levels. Data are shown as mean ± SD, n = 4, ***p

    Journal: Frontiers in Genetics

    Article Title: RNase H1 Regulates Mitochondrial Transcription and Translation via the Degradation of 7S RNA

    doi: 10.3389/fgene.2019.01393

    Figure Lengend Snippet: RNASEH1 mutations, transcript, and protein levels. (A) Domains ofhuman RNAse H1 protein (MTS, mitochondrial targeting sequence; HBD, hybrid binding domain; CD, connection domain; catalytic domain). RNase H1 protein sequences from representative species, H. sapiens (Hs, NP_002927) M. musculus (Mm, NP_035405), B. taurus (Bt, NP_001039970), G. gallus (Gg, NP_990329), X. tropicalis (Xt, NP_001096299), D. rerio (Dr, NP_001002659), C. intestinalis (Ci, F6QPH0), D. melanogaster (Dm, NP_995777), C. elegans (Ce, NP_001040786), S. cerevisiae (Sc, Q04740), were extracted from the database and aligned using ClustalW2. Conserved residues found mutated in the patient in exon 4 are boxed in red, while residues in the active site and interacting with DNA or RNA are boxed in yellow and green, respectively. Positions of β strands are marked by blue arrows. (B) Human RNase H1 crystal structure (PDB ID 2QK9) 18 bp DNA(cyan): RNA (magenta) hybrid is shown respectively. Residues in the active site are colored in yellow. Residues Trp 164 (green) and Val 142 , or the mutated variant Ile 142 (red), are shown as sticks. (C) RNASEH1 transcript levels in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium assessed by qPCR (probe Hs00268000_m1) and normalized to GAPDH transcript levels. Data are shown as mean ± SD, n = 4, ***p

    Article Snippet: In order to investigate if the presence of a nonsense mutation was triggering nonsense-mediated decay, we checked RNASEH1 transcript levels in human control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium with probe Hs00268000_m1, spanning exons 7-8 ( ).

    Techniques: Sequencing, Binding Assay, Variant Assay, Real-time Polymerase Chain Reaction

    Mitochondrial transcript levels in mutant RNASEH1 fibroblasts. Mitochondrial transcript levels in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium, assessed by qPCR and normalized to GAPDH transcript levels. Analyzed transcripts included the non-coding 7S RNA (MT-7S), the two ribosomal RNAs MT-RNR1 (12S rRNA) and MT-RNR2 (16S rRNA), three complex IV protein mRNAs (MT-CO1, MT-CO2 and MT-CO3), three complex I protein mRNAs (MT-ND1, MT-ND5 and MT-ND6), one complex III protein mRNA (MT-CYB), and one complex V protein mRNA (MT-ATP6). Data are shown as mean ± SD, n = 4, Student’s unpaired two-tail t-test, **p

    Journal: Frontiers in Genetics

    Article Title: RNase H1 Regulates Mitochondrial Transcription and Translation via the Degradation of 7S RNA

    doi: 10.3389/fgene.2019.01393

    Figure Lengend Snippet: Mitochondrial transcript levels in mutant RNASEH1 fibroblasts. Mitochondrial transcript levels in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium, assessed by qPCR and normalized to GAPDH transcript levels. Analyzed transcripts included the non-coding 7S RNA (MT-7S), the two ribosomal RNAs MT-RNR1 (12S rRNA) and MT-RNR2 (16S rRNA), three complex IV protein mRNAs (MT-CO1, MT-CO2 and MT-CO3), three complex I protein mRNAs (MT-ND1, MT-ND5 and MT-ND6), one complex III protein mRNA (MT-CYB), and one complex V protein mRNA (MT-ATP6). Data are shown as mean ± SD, n = 4, Student’s unpaired two-tail t-test, **p

    Article Snippet: In order to investigate if the presence of a nonsense mutation was triggering nonsense-mediated decay, we checked RNASEH1 transcript levels in human control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium with probe Hs00268000_m1, spanning exons 7-8 ( ).

    Techniques: Mutagenesis, Real-time Polymerase Chain Reaction

    Mitochondrial translation in mutant RNASEH1 fibroblasts. (A) Western blot analysis of mitochondrial proteins involved in mitochondrial RNA metabolism (RNA metab.) and mitochondrial large (mtLSU) and small (mtSSU) ribosomal subunits in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium. GAPDH was used as loading control. GAPDH is from the same blot as Figure1D . (B) Quantification of the Western blots shown in (A) normalized to GAPDH levels. Data are shown as mean ± SD, Student’s unpaired two-tail t-test, *p

    Journal: Frontiers in Genetics

    Article Title: RNase H1 Regulates Mitochondrial Transcription and Translation via the Degradation of 7S RNA

    doi: 10.3389/fgene.2019.01393

    Figure Lengend Snippet: Mitochondrial translation in mutant RNASEH1 fibroblasts. (A) Western blot analysis of mitochondrial proteins involved in mitochondrial RNA metabolism (RNA metab.) and mitochondrial large (mtLSU) and small (mtSSU) ribosomal subunits in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium. GAPDH was used as loading control. GAPDH is from the same blot as Figure1D . (B) Quantification of the Western blots shown in (A) normalized to GAPDH levels. Data are shown as mean ± SD, Student’s unpaired two-tail t-test, *p

    Article Snippet: In order to investigate if the presence of a nonsense mutation was triggering nonsense-mediated decay, we checked RNASEH1 transcript levels in human control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium with probe Hs00268000_m1, spanning exons 7-8 ( ).

    Techniques: Mutagenesis, Western Blot

    Mitochondrial DNA maintenance in mutant RNASEH1 fibroblasts. (A) Western blot analysis of representative components of the mitochondrial OxPhos complexes I-V in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium. GAPDH was used as loading control. (B) Oxygen consumption ( I O2 ) measurements in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium. Values of basal and maximal respiration along with ATP production-dependent, proton leak respiration, and spare capacity are presented. Data are shown as mean ± SD, n = 4, Student’s unpaired two-tail t-test, **p

    Journal: Frontiers in Genetics

    Article Title: RNase H1 Regulates Mitochondrial Transcription and Translation via the Degradation of 7S RNA

    doi: 10.3389/fgene.2019.01393

    Figure Lengend Snippet: Mitochondrial DNA maintenance in mutant RNASEH1 fibroblasts. (A) Western blot analysis of representative components of the mitochondrial OxPhos complexes I-V in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium. GAPDH was used as loading control. (B) Oxygen consumption ( I O2 ) measurements in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium. Values of basal and maximal respiration along with ATP production-dependent, proton leak respiration, and spare capacity are presented. Data are shown as mean ± SD, n = 4, Student’s unpaired two-tail t-test, **p

    Article Snippet: In order to investigate if the presence of a nonsense mutation was triggering nonsense-mediated decay, we checked RNASEH1 transcript levels in human control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium with probe Hs00268000_m1, spanning exons 7-8 ( ).

    Techniques: Mutagenesis, Western Blot

    m 5 C mRNA methylation is enriched at transcriptionally active sites with DNA damage. a U2OS-TRE cells transfected with TA-KR/TA-Cherry/tetR-KR/tetR-Cherry plasmids were exposed to light for 30 min for KR activation and allowed to recover for 1 h before harvest (scale bar: 10 μm). Quantification of frequency of cells in 500 cells with m 5 C foci from three independent experiments, mean ± SD (upper right). Fold increase of m 5 C mean intensity = mean intensity of m 5 C at TA-KR/mean intensity of background ( n = 20, mean ± SD) (lower right). b U2OS-TRE cells were transfected with TA-KR/TA-Cherry to induce local oxidative damage or for the control condition. Cells were then stained for m 5 C with four different anti-m 5 C antibodies. Frequency of m 5 C-positive cells in 500 cells was quantified ( n = 3, mean ± SD). c U2OS-TRE cells transfected with TA-KR were digested with RNaseH1, RNaseA, or DNase I and stained for m 5 C quantification (scale bar: 10 μm). d The mRNA from Flp-in 293 cells treated with or without 2 mM H 2 O 2 for 40 min was used for m 5 C measurement via dot blot. Quantification of m 5 C levels (mean ± SD) from three independent experiments normalized with Ctrl and methylene blue is shown. e 32 P-labeled mRNA monophosphate nucleosides were run on 2D gels for 2D-TLC analysis. In vitro-transcribed 4B mRNA with or without m 5 C was run in parallel. Representative images from three sets of independent experiments are shown with arrows showing the directions of each solvent run. Position of each nucleotide and m 5 C are labeled (Left). f 32 P-labeled mRNA monophosphate nucleosides from U2OS cells with or without 2 mM H 2 O 2 for 40 min were run on 2D gels for 2D-TLC analysis. Representative images from three sets of independent experiments. Associated quantification of relative increase in m 5 C in peroxide-treated cells compared to control, normalized to nucleotide C (right). Statistical analysis was performed with the unpaired two tailed Student’s t -test. * p

    Journal: Nature Communications

    Article Title: m5C modification of mRNA serves a DNA damage code to promote homologous recombination

    doi: 10.1038/s41467-020-16722-7

    Figure Lengend Snippet: m 5 C mRNA methylation is enriched at transcriptionally active sites with DNA damage. a U2OS-TRE cells transfected with TA-KR/TA-Cherry/tetR-KR/tetR-Cherry plasmids were exposed to light for 30 min for KR activation and allowed to recover for 1 h before harvest (scale bar: 10 μm). Quantification of frequency of cells in 500 cells with m 5 C foci from three independent experiments, mean ± SD (upper right). Fold increase of m 5 C mean intensity = mean intensity of m 5 C at TA-KR/mean intensity of background ( n = 20, mean ± SD) (lower right). b U2OS-TRE cells were transfected with TA-KR/TA-Cherry to induce local oxidative damage or for the control condition. Cells were then stained for m 5 C with four different anti-m 5 C antibodies. Frequency of m 5 C-positive cells in 500 cells was quantified ( n = 3, mean ± SD). c U2OS-TRE cells transfected with TA-KR were digested with RNaseH1, RNaseA, or DNase I and stained for m 5 C quantification (scale bar: 10 μm). d The mRNA from Flp-in 293 cells treated with or without 2 mM H 2 O 2 for 40 min was used for m 5 C measurement via dot blot. Quantification of m 5 C levels (mean ± SD) from three independent experiments normalized with Ctrl and methylene blue is shown. e 32 P-labeled mRNA monophosphate nucleosides were run on 2D gels for 2D-TLC analysis. In vitro-transcribed 4B mRNA with or without m 5 C was run in parallel. Representative images from three sets of independent experiments are shown with arrows showing the directions of each solvent run. Position of each nucleotide and m 5 C are labeled (Left). f 32 P-labeled mRNA monophosphate nucleosides from U2OS cells with or without 2 mM H 2 O 2 for 40 min were run on 2D gels for 2D-TLC analysis. Representative images from three sets of independent experiments. Associated quantification of relative increase in m 5 C in peroxide-treated cells compared to control, normalized to nucleotide C (right). Statistical analysis was performed with the unpaired two tailed Student’s t -test. * p

    Article Snippet: For RNaseA treatment: after heat treatment, cells were incubated with 100 μg/mL RNaseA in 100 μL RNase digestion buffer (5 mM EDTA, 300 mM NaCl, 10 mM Tris-HCl, pH 7.5) at room temperature for 25 min. For RNaseH1 treatment, the cells were incubated with 15 U RNaseH1 (Cat#: EN0201, ThermoFisher Scientific) in 100 μL reaction buffer (200 mM Tris-HCl, pH 7.8, 400 mM KCl, 80 mM MgCl2 , 10 mM DTT) at room temperature for 25 min. For DNase I treatment, cells were incubated with 20 U (1 μL) DNase I in 100 μL buffer (10 mM Tris-HCl, 2.5 mM MgCl2 , 0.5 mM CaCl2 , pH 7.5) at 37 °C for 30 min followed by heat treatment.

    Techniques: Methylation, Transfection, Activation Assay, Staining, Dot Blot, Labeling, Thin Layer Chromatography, In Vitro, Two Tailed Test

    Overexpression of rnh1 relieves replication pausing. A–D , 2DNAGE of four restriction fragments of Drosophila S2 cells mtDNA, probed as indicated, in material from control cells and cells overexpressing RNase H1 in the form of epitope-tagged RNase H1-V5 (denoted OE ), both treated with 500 μ m CuSO 4 for 48 h to induce expression. E , schematic map of Drosophila mtDNA, as also shown in Fig. 8 , indicating the location of relevant restriction sites ( open circles ), mTTF-binding sites (bs1 and bs2; filled circles ), the noncoding region ( bold ), and the probes used. The open arrowhead marks the location and direction of replication initiation (see Ref. 40 ). The directions of first- and second-dimension electrophoresis in all gels are as indicated by the arrows . The images show relatively low exposures to reveal fine details of the arcs of RIs.

    Journal: The Journal of Biological Chemistry

    Article Title: RNase H1 promotes replication fork progression through oppositely transcribed regions of Drosophila mitochondrial DNA

    doi: 10.1074/jbc.RA118.007015

    Figure Lengend Snippet: Overexpression of rnh1 relieves replication pausing. A–D , 2DNAGE of four restriction fragments of Drosophila S2 cells mtDNA, probed as indicated, in material from control cells and cells overexpressing RNase H1 in the form of epitope-tagged RNase H1-V5 (denoted OE ), both treated with 500 μ m CuSO 4 for 48 h to induce expression. E , schematic map of Drosophila mtDNA, as also shown in Fig. 8 , indicating the location of relevant restriction sites ( open circles ), mTTF-binding sites (bs1 and bs2; filled circles ), the noncoding region ( bold ), and the probes used. The open arrowhead marks the location and direction of replication initiation (see Ref. 40 ). The directions of first- and second-dimension electrophoresis in all gels are as indicated by the arrows . The images show relatively low exposures to reveal fine details of the arcs of RIs.

    Article Snippet: To establish cell clones stably expressing V5-tagged RNase H1 and variants, pCoBlast (Thermo Fisher Scientific) was included in transfections.

    Techniques: Over Expression, Expressing, Binding Assay, Electrophoresis

    Subcellular localization of epitope-tagged RNase H1. A , immunocytochemistry of cells transiently transfected with RNase H1-V5, probed for the V5 epitope tag ( red ), Cox4 ( green ), and DAPI ( blue ), showing examples of the three types of intracellular distribution of V5-tagged RNase H1: nucleus and mitochondria ( i ), mitochondria only ( ii ), and nucleus only ( iii ). B , subcellular distribution of RNase H1-V5 in 100 transfected cells as indicated (mean of three experiments, error bars denote S.D.). C , Western blots of subcellular fractions from cells transfected with RNase H1-V5, highly enriched for nuclei ( nuc ) or mitochondria ( mt ) as indicated, probed simultaneously for V5 and for the markers indicated. M , molecular mass markers.

    Journal: The Journal of Biological Chemistry

    Article Title: RNase H1 promotes replication fork progression through oppositely transcribed regions of Drosophila mitochondrial DNA

    doi: 10.1074/jbc.RA118.007015

    Figure Lengend Snippet: Subcellular localization of epitope-tagged RNase H1. A , immunocytochemistry of cells transiently transfected with RNase H1-V5, probed for the V5 epitope tag ( red ), Cox4 ( green ), and DAPI ( blue ), showing examples of the three types of intracellular distribution of V5-tagged RNase H1: nucleus and mitochondria ( i ), mitochondria only ( ii ), and nucleus only ( iii ). B , subcellular distribution of RNase H1-V5 in 100 transfected cells as indicated (mean of three experiments, error bars denote S.D.). C , Western blots of subcellular fractions from cells transfected with RNase H1-V5, highly enriched for nuclei ( nuc ) or mitochondria ( mt ) as indicated, probed simultaneously for V5 and for the markers indicated. M , molecular mass markers.

    Article Snippet: To establish cell clones stably expressing V5-tagged RNase H1 and variants, pCoBlast (Thermo Fisher Scientific) was included in transfections.

    Techniques: Immunocytochemistry, Transfection, Western Blot

    Subcellular targeting of RNase H1 variants. A , intracellular localization of RNase H1-V5 variants in cultures of stably transfected cells exemplified in B . M1V and M16V, N-terminal methionine variants (see Fig. S2 A ); ΔNLS, with the putative nuclear localization signal deleted (see Fig. S2 C ). C , intracellular localization of RNase H1-V5 in cells synchronized in G1 and G2 (see FACS profiles in Fig. S2 E ). All plotted values are means of three experiments. Error bars denote S.D. nuc , nuclei; mt , mitochondria.

    Journal: The Journal of Biological Chemistry

    Article Title: RNase H1 promotes replication fork progression through oppositely transcribed regions of Drosophila mitochondrial DNA

    doi: 10.1074/jbc.RA118.007015

    Figure Lengend Snippet: Subcellular targeting of RNase H1 variants. A , intracellular localization of RNase H1-V5 variants in cultures of stably transfected cells exemplified in B . M1V and M16V, N-terminal methionine variants (see Fig. S2 A ); ΔNLS, with the putative nuclear localization signal deleted (see Fig. S2 C ). C , intracellular localization of RNase H1-V5 in cells synchronized in G1 and G2 (see FACS profiles in Fig. S2 E ). All plotted values are means of three experiments. Error bars denote S.D. nuc , nuclei; mt , mitochondria.

    Article Snippet: To establish cell clones stably expressing V5-tagged RNase H1 and variants, pCoBlast (Thermo Fisher Scientific) was included in transfections.

    Techniques: Stable Transfection, Transfection, FACS