Structured Review

Thermo Fisher rnaseh1
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.
Rnaseh1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) 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, Hemagglutination 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, Hemagglutination 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

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    Thermo Fisher rnaseh1
    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.
    Rnaseh1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnaseh1/product/Thermo Fisher
    Average 77 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    rnaseh1 - by Bioz Stars, 2020-01
    77/100 stars
      Buy from Supplier

    86
    Thermo Fisher gene exp rnaseh1 mm00488036 m1
    RNase H1 Knockdown Suppresses LNA-ASO Toxicity  In Vitro (A) Mouse 3T3 fibroblast cells were transfected with a siRNA against RNase H1 (light gray bars) and a control (ctr.) siRNA (dark gray bars) at 10 nM, respectively, using Lipofectamine 2000. 24 hr later, cells were transfected with the hepatotoxic LNA41 or LNA37 at 30 nM using Lipofectamine 2000. Caspase 3/7 activity was measured 24 hr later. (B) siRNA transfection was performed as in (A) and cells were harvested 48 hr later for assessing RNase H1 knockdown by qPCR. Results in (A) and (B) are shown as a percentage change relative to UTCs. Data (n = 3) are mean ± SD.
    Gene Exp Rnaseh1 Mm00488036 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gene exp rnaseh1 mm00488036 m1/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    gene exp rnaseh1 mm00488036 m1 - by Bioz Stars, 2020-01
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    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.

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0071006

    Figure Lengend 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.

    Article Snippet: The full length human RNase H1, H2, and P32 cDNAs (GenBank accession numbers NM-002936, NM-006397, and NM-001212, respectively) were used to construct the plasmids with N-terminal Flag- or C-terminal HA-tag in pcDNA3.1 vector (Invitrogen) for transient expression or creation of stable cell lines.

    Techniques: Polymerase Chain Reaction, Derivative Assay, Hemagglutination 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.

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0071006

    Figure Lengend 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.

    Article Snippet: The full length human RNase H1, H2, and P32 cDNAs (GenBank accession numbers NM-002936, NM-006397, and NM-001212, respectively) were used to construct the plasmids with N-terminal Flag- or C-terminal HA-tag in pcDNA3.1 vector (Invitrogen) for transient expression or creation of stable cell lines.

    Techniques: 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.

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0071006

    Figure Lengend 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.

    Article Snippet: The full length human RNase H1, H2, and P32 cDNAs (GenBank accession numbers NM-002936, NM-006397, and NM-001212, respectively) were used to construct the plasmids with N-terminal Flag- or C-terminal HA-tag in pcDNA3.1 vector (Invitrogen) for transient expression or creation of stable cell lines.

    Techniques: 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.

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0071006

    Figure Lengend 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.

    Article Snippet: The full length human RNase H1, H2, and P32 cDNAs (GenBank accession numbers NM-002936, NM-006397, and NM-001212, respectively) were used to construct the plasmids with N-terminal Flag- or C-terminal HA-tag in pcDNA3.1 vector (Invitrogen) for transient expression or creation of stable cell lines.

    Techniques: 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.

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0071006

    Figure Lengend 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.

    Article Snippet: The full length human RNase H1, H2, and P32 cDNAs (GenBank accession numbers NM-002936, NM-006397, and NM-001212, respectively) were used to construct the plasmids with N-terminal Flag- or C-terminal HA-tag in pcDNA3.1 vector (Invitrogen) for transient expression or creation of stable cell lines.

    Techniques: 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.

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0071006

    Figure Lengend 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.

    Article Snippet: The full length human RNase H1, H2, and P32 cDNAs (GenBank accession numbers NM-002936, NM-006397, and NM-001212, respectively) were used to construct the plasmids with N-terminal Flag- or C-terminal HA-tag in pcDNA3.1 vector (Invitrogen) for transient expression or creation of stable cell lines.

    Techniques: Western Blot, Immunoprecipitation, Expressing, Stable Transfection, Transformation Assay, Selection, Binding Assay, Hemagglutination 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

    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

    RNase H1 Knockdown Suppresses LNA-ASO Toxicity In Vitro (A) Mouse 3T3 fibroblast cells were transfected with a siRNA against RNase H1 (light gray bars) and a control (ctr.) siRNA (dark gray bars) at 10 nM, respectively, using Lipofectamine 2000. 24 hr later, cells were transfected with the hepatotoxic LNA41 or LNA37 at 30 nM using Lipofectamine 2000. Caspase 3/7 activity was measured 24 hr later. (B) siRNA transfection was performed as in (A) and cells were harvested 48 hr later for assessing RNase H1 knockdown by qPCR. Results in (A) and (B) are shown as a percentage change relative to UTCs. Data (n = 3) are mean ± SD.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: A Sensitive In Vitro Approach to Assess the Hybridization-Dependent Toxic Potential of High Affinity Gapmer Oligonucleotides

    doi: 10.1016/j.omtn.2017.11.004

    Figure Lengend Snippet: RNase H1 Knockdown Suppresses LNA-ASO Toxicity In Vitro (A) Mouse 3T3 fibroblast cells were transfected with a siRNA against RNase H1 (light gray bars) and a control (ctr.) siRNA (dark gray bars) at 10 nM, respectively, using Lipofectamine 2000. 24 hr later, cells were transfected with the hepatotoxic LNA41 or LNA37 at 30 nM using Lipofectamine 2000. Caspase 3/7 activity was measured 24 hr later. (B) siRNA transfection was performed as in (A) and cells were harvested 48 hr later for assessing RNase H1 knockdown by qPCR. Results in (A) and (B) are shown as a percentage change relative to UTCs. Data (n = 3) are mean ± SD.

    Article Snippet: The primer probes for mouse Myd88 , RNase H1 , and GAPDH were Mm00440338_m1, Mm00488036_m1, and Mm99999915_g1 (all from Thermo Fisher Scientific), respectively.

    Techniques: Allele-specific Oligonucleotide, In Vitro, Transfection, Activity Assay, Real-time Polymerase Chain Reaction

    RNase H1 Knockdown Suppresses LNA-ASO Toxicity  In Vitro (A) Mouse 3T3 fibroblast cells were transfected with a siRNA against RNase H1 (light gray bars) and a control (ctr.) siRNA (dark gray bars) at 10 nM, respectively, using Lipofectamine 2000. 24 hr later, cells were transfected with the hepatotoxic LNA41 or LNA37 at 30 nM using Lipofectamine 2000. Caspase 3/7 activity was measured 24 hr later. (B) siRNA transfection was performed as in (A) and cells were harvested 48 hr later for assessing RNase H1 knockdown by qPCR. Results in (A) and (B) are shown as a percentage change relative to UTCs. Data (n = 3) are mean ± SD.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: A Sensitive In Vitro Approach to Assess the Hybridization-Dependent Toxic Potential of High Affinity Gapmer Oligonucleotides

    doi: 10.1016/j.omtn.2017.11.004

    Figure Lengend Snippet: RNase H1 Knockdown Suppresses LNA-ASO Toxicity In Vitro (A) Mouse 3T3 fibroblast cells were transfected with a siRNA against RNase H1 (light gray bars) and a control (ctr.) siRNA (dark gray bars) at 10 nM, respectively, using Lipofectamine 2000. 24 hr later, cells were transfected with the hepatotoxic LNA41 or LNA37 at 30 nM using Lipofectamine 2000. Caspase 3/7 activity was measured 24 hr later. (B) siRNA transfection was performed as in (A) and cells were harvested 48 hr later for assessing RNase H1 knockdown by qPCR. Results in (A) and (B) are shown as a percentage change relative to UTCs. Data (n = 3) are mean ± SD.

    Article Snippet: The primer probes for mouse Myd88 , RNase H1 , and GAPDH were Mm00440338_m1, Mm00488036_m1, and Mm99999915_g1 (all from Thermo Fisher Scientific), respectively.

    Techniques: Allele-specific Oligonucleotide, In Vitro, Transfection, Activity Assay, Real-time Polymerase Chain Reaction