Structured Review

Roche rnase a
RNA–DNA hybrid formation at a (CGG)39 · (CCG)39 FRAXA template. When the template DNA is transcribed with T7 RNA polymerase, heterogeneous RNA is produced generating a smear (Transcription lane). Treatment with RNase H alone which is specific to RNA base paired to template DNA digests only RNA that is base paired to its template DNA. Treatment with RNase A, which is specific to single-stranded RNA, digests all free, single-stranded RNA leaving template DNA and RNA:DNA hybrid structures. Note that RNA–DNA hybrids migrate more slowly than supercoiled DNA (as indicated schematically, RNA is in light blue). Hybrid structures generate a smear due to their heterogeneous sizes. With a larger RNA component, the DNA is open to a greater degree (more relaxed), hence migration is closer to open circular DNA. Treatment of the hybrids with RNase H along with <t>RNase</t> A removes any hybrids formed as well as transcript generated in the transcription reaction leaving only input template DNA. When transcription followed by RNase H or RNase A treatment alone or in combination is performed on an empty Bluescript vector [pBlueKS(+)], there is negligible hybrid formation.
Rnase A, supplied by Roche, used in various techniques. Bioz Stars score: 98/100, based on 60 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/rnase a/product/Roche
Average 98 stars, based on 60 article reviews
Price from $9.99 to $1999.99
rnase a - by Bioz Stars, 2020-01
98/100 stars

Images

1) Product Images from "Determinants of R-loop formation at convergent bidirectionally transcribed trinucleotide repeats"

Article Title: Determinants of R-loop formation at convergent bidirectionally transcribed trinucleotide repeats

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq935

RNA–DNA hybrid formation at a (CGG)39 · (CCG)39 FRAXA template. When the template DNA is transcribed with T7 RNA polymerase, heterogeneous RNA is produced generating a smear (Transcription lane). Treatment with RNase H alone which is specific to RNA base paired to template DNA digests only RNA that is base paired to its template DNA. Treatment with RNase A, which is specific to single-stranded RNA, digests all free, single-stranded RNA leaving template DNA and RNA:DNA hybrid structures. Note that RNA–DNA hybrids migrate more slowly than supercoiled DNA (as indicated schematically, RNA is in light blue). Hybrid structures generate a smear due to their heterogeneous sizes. With a larger RNA component, the DNA is open to a greater degree (more relaxed), hence migration is closer to open circular DNA. Treatment of the hybrids with RNase H along with RNase A removes any hybrids formed as well as transcript generated in the transcription reaction leaving only input template DNA. When transcription followed by RNase H or RNase A treatment alone or in combination is performed on an empty Bluescript vector [pBlueKS(+)], there is negligible hybrid formation.
Figure Legend Snippet: RNA–DNA hybrid formation at a (CGG)39 · (CCG)39 FRAXA template. When the template DNA is transcribed with T7 RNA polymerase, heterogeneous RNA is produced generating a smear (Transcription lane). Treatment with RNase H alone which is specific to RNA base paired to template DNA digests only RNA that is base paired to its template DNA. Treatment with RNase A, which is specific to single-stranded RNA, digests all free, single-stranded RNA leaving template DNA and RNA:DNA hybrid structures. Note that RNA–DNA hybrids migrate more slowly than supercoiled DNA (as indicated schematically, RNA is in light blue). Hybrid structures generate a smear due to their heterogeneous sizes. With a larger RNA component, the DNA is open to a greater degree (more relaxed), hence migration is closer to open circular DNA. Treatment of the hybrids with RNase H along with RNase A removes any hybrids formed as well as transcript generated in the transcription reaction leaving only input template DNA. When transcription followed by RNase H or RNase A treatment alone or in combination is performed on an empty Bluescript vector [pBlueKS(+)], there is negligible hybrid formation.

Techniques Used: Produced, Migration, Generated, Plasmid Preparation

Quantification of relative RNA–DNA hybrid formation at increasing repeat lengths. ( A ) In vitro transcription reactions were performed on FRAXA plasmids bearing repeat tracts of (CGG)17 · (CCG)17, (CGG)39 · (CCG)39 and (CGG)53 · (CCG)53 following which samples were treated with RNase A or A+H. Hybrid formation was quantified by densitometry analysis using image quant by measuring the proportion of products that migrate between open circular and supercoiled position (indicated by ‘R’) divided by the total products below open circular including supercoiled. RNase A+H treated samples were used to determine the position of the supercoiled DNA for each repeat length. To the left of the graph is a representative gel used to quantify relative hybrid formation. The RNA indicated in the graph represents the RNA component bound in the RNA:DNA hybrid. Error bars are derived from three separate experiments ( N =3). Using the t -test to compare R-loop formation revealed a significant difference between the 17 and 53 rCGG R-loop ( P =0.0413) as well as the 17 and 53 rCCG R-loop ( P =0.0092). ( B ) The same analysis was performed as in (A) but for SCA1 plasmids bearing repeat tracts of (CAG)30 · (CTG)30, (CAG) 49 · (CTG)49 and (CAG)74 · (CTG)74. Using the t -test to compare R-loop formation between each of the (CAG) · (CTG) repeat lengths did not reveal any statistically significant differences.
Figure Legend Snippet: Quantification of relative RNA–DNA hybrid formation at increasing repeat lengths. ( A ) In vitro transcription reactions were performed on FRAXA plasmids bearing repeat tracts of (CGG)17 · (CCG)17, (CGG)39 · (CCG)39 and (CGG)53 · (CCG)53 following which samples were treated with RNase A or A+H. Hybrid formation was quantified by densitometry analysis using image quant by measuring the proportion of products that migrate between open circular and supercoiled position (indicated by ‘R’) divided by the total products below open circular including supercoiled. RNase A+H treated samples were used to determine the position of the supercoiled DNA for each repeat length. To the left of the graph is a representative gel used to quantify relative hybrid formation. The RNA indicated in the graph represents the RNA component bound in the RNA:DNA hybrid. Error bars are derived from three separate experiments ( N =3). Using the t -test to compare R-loop formation revealed a significant difference between the 17 and 53 rCGG R-loop ( P =0.0413) as well as the 17 and 53 rCCG R-loop ( P =0.0092). ( B ) The same analysis was performed as in (A) but for SCA1 plasmids bearing repeat tracts of (CAG)30 · (CTG)30, (CAG) 49 · (CTG)49 and (CAG)74 · (CTG)74. Using the t -test to compare R-loop formation between each of the (CAG) · (CTG) repeat lengths did not reveal any statistically significant differences.

Techniques Used: In Vitro, Derivative Assay, CTG Assay

Effect of trinucleotide repeat interruptions on RNA–DNA hybrid formation. ( A ) In vitro transcription followed by RNase A or A + H treatment was performed with either pure (39p) FRAXA plasmids (CGG)39 · (CCG)39 or interrupted plasmids (39i) [(CGG)9(AGG) (CGG)9(AGG)(CGG)9(AGG)(CGG)9] · [(CCG)9(CCT) (CCG)9(CCT)(CCG)9(CCT)(CCG)9] as indicated. Repeat tract configurations are schematically presented where hollow dots are the CGG repeat units and the filled dots are the AGG interruptions. R-loops are indicated as ‘R’. ( B ) In vitro transcription followed by RNase A or A+H treatment was performed with either pure (49p) SCA1 plasmids (CAG)49 · (CTG)49 or interrupted plasmids (44i) [(CAG)12(CAT)(CAG)(CAT)(CAG)12(CAT)(CAG)(CAT)(CAG)14] · [(CTG)14(ATG)(CTG)(ATG)(CTG)12(ATG)(CAG)(ATG)(CTG)12] as indicated schematically where hollow dots are the CAG repeat units and the filled dots are the CAT interruptions.
Figure Legend Snippet: Effect of trinucleotide repeat interruptions on RNA–DNA hybrid formation. ( A ) In vitro transcription followed by RNase A or A + H treatment was performed with either pure (39p) FRAXA plasmids (CGG)39 · (CCG)39 or interrupted plasmids (39i) [(CGG)9(AGG) (CGG)9(AGG)(CGG)9(AGG)(CGG)9] · [(CCG)9(CCT) (CCG)9(CCT)(CCG)9(CCT)(CCG)9] as indicated. Repeat tract configurations are schematically presented where hollow dots are the CGG repeat units and the filled dots are the AGG interruptions. R-loops are indicated as ‘R’. ( B ) In vitro transcription followed by RNase A or A+H treatment was performed with either pure (49p) SCA1 plasmids (CAG)49 · (CTG)49 or interrupted plasmids (44i) [(CAG)12(CAT)(CAG)(CAT)(CAG)12(CAT)(CAG)(CAT)(CAG)14] · [(CTG)14(ATG)(CTG)(ATG)(CTG)12(ATG)(CAG)(ATG)(CTG)12] as indicated schematically where hollow dots are the CAG repeat units and the filled dots are the CAT interruptions.

Techniques Used: In Vitro, CTG Assay

Identification of R-loop structures formed in expanded DM1 (CTG)130 · (CAG)130 plasmids using EM following in vitro transcription and treatment with RNase A and SSB protein. SSB proteins bind to the looped-out non-template DNA in an R-loop structure. Thus, each R-loop structure is visualized as a loop within the DNA template as indicated by black arrowheads. ( A ) R-loops formed by using SP6 RNA polymerase, producing an rCAG:dCTG hybrid. ( B ) R-loops formed by using T7 RNA polymerase, producing an rCUG:dCAG hybrid.
Figure Legend Snippet: Identification of R-loop structures formed in expanded DM1 (CTG)130 · (CAG)130 plasmids using EM following in vitro transcription and treatment with RNase A and SSB protein. SSB proteins bind to the looped-out non-template DNA in an R-loop structure. Thus, each R-loop structure is visualized as a loop within the DNA template as indicated by black arrowheads. ( A ) R-loops formed by using SP6 RNA polymerase, producing an rCAG:dCTG hybrid. ( B ) R-loops formed by using T7 RNA polymerase, producing an rCUG:dCAG hybrid.

Techniques Used: CTG Assay, In Vitro

RNA–DNA hybrid formation during in vitro transcription of trinucleotide repeat-containing plasmids. ( A ) In vitro transcription of SCA1 plasmid containing (CAG)74 · (CTG)74 and FRAXA plasmid containing (CGG)39 · (CCG)39 repeats in either direction using T3 or T7 RNA polymerase. The repeat sequence contained within the RNA produced and bound in the hybrid is indicated below the transcribed template. Samples following transcription were subsequently treated with either RNase A or A+H as indicated to observe hybrid formation. R-loops are indicated on the gel as ‘R’. ( B ) Exact same reactions and gel conditions as in (A) but transcription was performed in the presence of 3.5 µCi [α- 32 P]-rCTP. Gel was dried and exposed to X-ray film as outlined in ‘Materials and Methods’ section.
Figure Legend Snippet: RNA–DNA hybrid formation during in vitro transcription of trinucleotide repeat-containing plasmids. ( A ) In vitro transcription of SCA1 plasmid containing (CAG)74 · (CTG)74 and FRAXA plasmid containing (CGG)39 · (CCG)39 repeats in either direction using T3 or T7 RNA polymerase. The repeat sequence contained within the RNA produced and bound in the hybrid is indicated below the transcribed template. Samples following transcription were subsequently treated with either RNase A or A+H as indicated to observe hybrid formation. R-loops are indicated on the gel as ‘R’. ( B ) Exact same reactions and gel conditions as in (A) but transcription was performed in the presence of 3.5 µCi [α- 32 P]-rCTP. Gel was dried and exposed to X-ray film as outlined in ‘Materials and Methods’ section.

Techniques Used: In Vitro, Plasmid Preparation, CTG Assay, Sequencing, Produced

Effect of convergent simultaneous bidirectional or serial transcription on R-loop formation. ( A ) In vitro transcription of FRAXA template (CGG)39 · (CCG)39 with either T3 or T7 RNA polymerase alone (rCCG or rCGG, respectively), or simultaneously (rCCG with rCGG) or serially: rCCG transcription then phenol chloroform extraction followed by rCGG transcription (rCCG then rCGG), and vice versa (rCGG then rCCG). R-loops are indicated as ‘R’. Note that in the case of bidirectional or serial transcription, complementary RNA is produced forming dsRNA as indicated on the gel by ‘*’. These products are not present in transcription reactions occurring in one direction. ( B ) Same as in (A) except in vitro transcription reactions were performed on a DM1 (CAG)79 · (CTG)79 template. ( C ) EM analysis of convergent transcription reaction products from DM1 (CAG)79 · (CTG)79 templates. Samples were transcribed convergently using T3 and T7 RNA polymerase promoters simultaneously then the products were treated with RNAse A and prepared for EM as described in the ‘Materials and Methods’ section (rCUGand rCAG RNase A). Samples were also subjected to RNase H treatment along with RNase A for comparison (rCUG and rCAG RNase A, H). Transcription was also performed on the same template in a single direction for further comparison (rCUG RNase A). The products observed for each type of transcription reaction is shown as a percentage of the total number of molecules analyzed. At least 100 molecules were analyzed for each type of transcription reaction.
Figure Legend Snippet: Effect of convergent simultaneous bidirectional or serial transcription on R-loop formation. ( A ) In vitro transcription of FRAXA template (CGG)39 · (CCG)39 with either T3 or T7 RNA polymerase alone (rCCG or rCGG, respectively), or simultaneously (rCCG with rCGG) or serially: rCCG transcription then phenol chloroform extraction followed by rCGG transcription (rCCG then rCGG), and vice versa (rCGG then rCCG). R-loops are indicated as ‘R’. Note that in the case of bidirectional or serial transcription, complementary RNA is produced forming dsRNA as indicated on the gel by ‘*’. These products are not present in transcription reactions occurring in one direction. ( B ) Same as in (A) except in vitro transcription reactions were performed on a DM1 (CAG)79 · (CTG)79 template. ( C ) EM analysis of convergent transcription reaction products from DM1 (CAG)79 · (CTG)79 templates. Samples were transcribed convergently using T3 and T7 RNA polymerase promoters simultaneously then the products were treated with RNAse A and prepared for EM as described in the ‘Materials and Methods’ section (rCUGand rCAG RNase A). Samples were also subjected to RNase H treatment along with RNase A for comparison (rCUG and rCAG RNase A, H). Transcription was also performed on the same template in a single direction for further comparison (rCUG RNase A). The products observed for each type of transcription reaction is shown as a percentage of the total number of molecules analyzed. At least 100 molecules were analyzed for each type of transcription reaction.

Techniques Used: In Vitro, Produced, CTG Assay

Mechanism of RNA:DNA hybrid formation during in vitro transcription. ( A ) Schematic of the two possible mechanisms for R-loop formation. By the thread-back model, the nascent transcript (depicted in light blue) that has been ejected from the RNA polymerase re-anneals with the complementary, free DNA template strand (depicted in red) following the progression of the RNA polymerase (light blue, oval). When transcription occurs in the presence of RNAse A (dark blue) the nascent transcript is degraded when it is ejected from the RNA polymerase hence cannot form the hybrid. By the extended-hybrid model, the nascent transcript remains bound to the template DNA and resists becoming ejected from the RNA polymerase. When transcribed in the presence of RNase A, as the nascent transcript is protected by being bound to the template DNA it is not degraded hence hybrid formation is not ablated. ( B ) FRAXA plasmid (CGG)39 · (CCG)39 transcribed in either direction in the absence (−) or presence (+) of RNase A during the transcription reaction. All transcription reactions were subjected to further RNase A or A + H treatment to analyze hybrid formation. ( C ) Same experiment as in (B) performed with SCA1 plasmid containing a (CAG)74 · (CTG)74 repeat tract.
Figure Legend Snippet: Mechanism of RNA:DNA hybrid formation during in vitro transcription. ( A ) Schematic of the two possible mechanisms for R-loop formation. By the thread-back model, the nascent transcript (depicted in light blue) that has been ejected from the RNA polymerase re-anneals with the complementary, free DNA template strand (depicted in red) following the progression of the RNA polymerase (light blue, oval). When transcription occurs in the presence of RNAse A (dark blue) the nascent transcript is degraded when it is ejected from the RNA polymerase hence cannot form the hybrid. By the extended-hybrid model, the nascent transcript remains bound to the template DNA and resists becoming ejected from the RNA polymerase. When transcribed in the presence of RNase A, as the nascent transcript is protected by being bound to the template DNA it is not degraded hence hybrid formation is not ablated. ( B ) FRAXA plasmid (CGG)39 · (CCG)39 transcribed in either direction in the absence (−) or presence (+) of RNase A during the transcription reaction. All transcription reactions were subjected to further RNase A or A + H treatment to analyze hybrid formation. ( C ) Same experiment as in (B) performed with SCA1 plasmid containing a (CAG)74 · (CTG)74 repeat tract.

Techniques Used: In Vitro, Plasmid Preparation, CTG Assay

2) Product Images from "Evidence that Lin28 stimulates translation by recruiting RNA helicase A to polysomes"

Article Title: Evidence that Lin28 stimulates translation by recruiting RNA helicase A to polysomes

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq1350

The N- and C-terminal regions of RHA interact with Lin28. ( A ) Domain organization of human RHA protein. Double-stranded RNA binding domain I and II (dsRBD I and II), C-terminal domain rich in arginine-glycine-glycine (RGG) repeats and the Walker helicase motifs of the conserved DEAD-box RNA helicases are depicted. Numbers indicate corresponding amino acid residue. ( B ) GST pulldown results. HEK293 cell lysate containing Flag-Lin28 was incubated with bacterial lysate containing the indicated recombinant RHA domains or GST alone in the presence of RNase A, followed by GST pulldown assays. Left panel, anti-Flag and anti-PABP antibodies were used in the upper and lower blots, respectively. Input was 0.5% of the total amount of proteins used for each GST pulldown. Right panel, Coomassie staining determined comparable amounts of the recombinant proteins used in the GST pulldown assays. 1% of the input was loaded in each lane. Molecular size markers are on the right. ( C ) Flag-Lin28 and Flag-N300 were co-transfected into HEK293 cells. Co-IP was carried out in the presence of RNase A 24 h later using anti-Lin28 antibody to bring down Flag-Lin28 together with its associated proteins, followed by western blot analysis. Antibodies used in the western blot were anti-RHA (top two blots, note, this antibody recognizes both full-length RHA and Flag-N300), anti-NXF1 (third blot from top), and anti-Flag M2 (bottom blot). Total proteins (2%) used for each immunoprecipitation was loaded as input.
Figure Legend Snippet: The N- and C-terminal regions of RHA interact with Lin28. ( A ) Domain organization of human RHA protein. Double-stranded RNA binding domain I and II (dsRBD I and II), C-terminal domain rich in arginine-glycine-glycine (RGG) repeats and the Walker helicase motifs of the conserved DEAD-box RNA helicases are depicted. Numbers indicate corresponding amino acid residue. ( B ) GST pulldown results. HEK293 cell lysate containing Flag-Lin28 was incubated with bacterial lysate containing the indicated recombinant RHA domains or GST alone in the presence of RNase A, followed by GST pulldown assays. Left panel, anti-Flag and anti-PABP antibodies were used in the upper and lower blots, respectively. Input was 0.5% of the total amount of proteins used for each GST pulldown. Right panel, Coomassie staining determined comparable amounts of the recombinant proteins used in the GST pulldown assays. 1% of the input was loaded in each lane. Molecular size markers are on the right. ( C ) Flag-Lin28 and Flag-N300 were co-transfected into HEK293 cells. Co-IP was carried out in the presence of RNase A 24 h later using anti-Lin28 antibody to bring down Flag-Lin28 together with its associated proteins, followed by western blot analysis. Antibodies used in the western blot were anti-RHA (top two blots, note, this antibody recognizes both full-length RHA and Flag-N300), anti-NXF1 (third blot from top), and anti-Flag M2 (bottom blot). Total proteins (2%) used for each immunoprecipitation was loaded as input.

Techniques Used: RNA Binding Assay, Incubation, Recombinant, Staining, Transfection, Co-Immunoprecipitation Assay, Western Blot, Immunoprecipitation

C-terminus deletion reduces Lin28′s ability to interact with RHA. ( A ) Schematic of wild-type and mutant Lin28 protein. Numbers are in amino acids. ( B ) Flag-Lin28, Flag-Lin28ΔN, Flag-Lin28ΔC or empty vector were each transfected into HEK293 cells. Co-IP was carried out in the presence of RNase A using anti-Flag antibody. Resulting protein complexes were resolved by SDS–PAGE, followed by western blot analysis. Anti-RHA and polyclonal anti-Flag antibody were used in the top and bottom blots, respectively. Three percent of input was loaded. ( C ) Flag-Lin28ΔC was transfected into HEK293 cells. Cell lysate containing Flag-Lin28ΔC was incubated with bacterial lysate containing the indicated recombinant RHA domains or GST, followed by GST pulldown assays. Top panel, anti-Flag M2 antibody was used to detect Flag-Lin28ΔC. About 0.5% of the input was loaded in lane 1. Bottom panel, Coomassie staining of the recombinant proteins used in the GST pulldown assays. About 1% of the input was loaded in each lane. Molecular size markers are on the right.
Figure Legend Snippet: C-terminus deletion reduces Lin28′s ability to interact with RHA. ( A ) Schematic of wild-type and mutant Lin28 protein. Numbers are in amino acids. ( B ) Flag-Lin28, Flag-Lin28ΔN, Flag-Lin28ΔC or empty vector were each transfected into HEK293 cells. Co-IP was carried out in the presence of RNase A using anti-Flag antibody. Resulting protein complexes were resolved by SDS–PAGE, followed by western blot analysis. Anti-RHA and polyclonal anti-Flag antibody were used in the top and bottom blots, respectively. Three percent of input was loaded. ( C ) Flag-Lin28ΔC was transfected into HEK293 cells. Cell lysate containing Flag-Lin28ΔC was incubated with bacterial lysate containing the indicated recombinant RHA domains or GST, followed by GST pulldown assays. Top panel, anti-Flag M2 antibody was used to detect Flag-Lin28ΔC. About 0.5% of the input was loaded in lane 1. Bottom panel, Coomassie staining of the recombinant proteins used in the GST pulldown assays. About 1% of the input was loaded in each lane. Molecular size markers are on the right.

Techniques Used: Mutagenesis, Plasmid Preparation, Transfection, Co-Immunoprecipitation Assay, SDS Page, Western Blot, Incubation, Recombinant, Staining

Lin28 interacts with RHA in PA-1 cells. Lin28-containing protein complexes were immunoprecipitated in the presence of excess amounts of RNase A from PA-1 cells using anti-Lin28 or pre-immune IgG (as a negative control for non-specific binding). Co-IP complexes (lanes 1 and 2) and 3% input (lane 3) were resolved by SDS–PAGE, followed by western blot analysis using anti-RHA (top blot) and anti-NXF1 (bottom blot) antiboddies, respectively.
Figure Legend Snippet: Lin28 interacts with RHA in PA-1 cells. Lin28-containing protein complexes were immunoprecipitated in the presence of excess amounts of RNase A from PA-1 cells using anti-Lin28 or pre-immune IgG (as a negative control for non-specific binding). Co-IP complexes (lanes 1 and 2) and 3% input (lane 3) were resolved by SDS–PAGE, followed by western blot analysis using anti-RHA (top blot) and anti-NXF1 (bottom blot) antiboddies, respectively.

Techniques Used: Immunoprecipitation, Negative Control, Binding Assay, Co-Immunoprecipitation Assay, SDS Page, Western Blot

3) Product Images from "Genetic and Biochemical Assays Reveal a Key Role for Replication Restart Proteins in Group II Intron Retrohoming"

Article Title: Genetic and Biochemical Assays Reveal a Key Role for Replication Restart Proteins in Group II Intron Retrohoming

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1003469

E. coli  extract assay for bottom-strand (cDNA) and top-strand DNA synthesis. (A) Time courses. Group II intron RNPs and labeled DNA substrates (73 bp) containing the Ll.LtrB-insertion site (ligated E1–E2 sequence) were incubated with  E. coli  HMS174(DE3) extract in the presence of 1 mM dNTPs, 1.5 mM ATP, and an ATP-regenerating system (phosphoenolpyruvate+pyruvate kinase) at 37°C. The DNA substrates were labeled at the 5′ end of either the top (T) or bottom (B) strands to separately assay top- and bottom-strand DNA synthesis. After terminating portions of the reaction at the indicated times, samples were split into halves, which were incubated without or with RNases A+H, and the products were analyzed in a denaturing 6% polyacrylamide gel, which was dried and scanned with a PhosphorImager. RNase-sensitive top-strand products contain the reverse-spliced intron RNA. Schematics below the gels depict bottom- and top-strand synthesis on the DNA substrates (intron and exons not drawn to scale; star indicates 5′  32 P-label). (B) Primer extension analysis. DNA products synthesized in a time course were digested with RNase A+H, purified in a 1% agarose gel (0.85–1.2 kb gel slice), and analyzed by primer extension using 5′ -labeled primers to detect bottom-strand cDNAs (primer FB); the top-strand 5′-intron-integration junction (primer 5T); and top-strand DNAs (primer FT). Major products are diagrammed below the gel. (C) Requirements for bottom- and top-strand DNA synthesis. Reactions with the indicated components were incubated at 37°C for 30 min and then processed and analyzed in a denaturing 6% polyacrylamide gel, as described above. (D) Bottom- and top-strand products obtained with RNPs containing wild-type LtrA protein or an RT-deficient mutant LtrA (RT − ; YADD motif changed to YAAA). For simplicity, the bottom part of the gel with the labeled DNA substrate (S) is shown only for panel D. Asterisks indicate gels bands of the size expected for full-length bottom- and top-strand products.
Figure Legend Snippet: E. coli extract assay for bottom-strand (cDNA) and top-strand DNA synthesis. (A) Time courses. Group II intron RNPs and labeled DNA substrates (73 bp) containing the Ll.LtrB-insertion site (ligated E1–E2 sequence) were incubated with E. coli HMS174(DE3) extract in the presence of 1 mM dNTPs, 1.5 mM ATP, and an ATP-regenerating system (phosphoenolpyruvate+pyruvate kinase) at 37°C. The DNA substrates were labeled at the 5′ end of either the top (T) or bottom (B) strands to separately assay top- and bottom-strand DNA synthesis. After terminating portions of the reaction at the indicated times, samples were split into halves, which were incubated without or with RNases A+H, and the products were analyzed in a denaturing 6% polyacrylamide gel, which was dried and scanned with a PhosphorImager. RNase-sensitive top-strand products contain the reverse-spliced intron RNA. Schematics below the gels depict bottom- and top-strand synthesis on the DNA substrates (intron and exons not drawn to scale; star indicates 5′ 32 P-label). (B) Primer extension analysis. DNA products synthesized in a time course were digested with RNase A+H, purified in a 1% agarose gel (0.85–1.2 kb gel slice), and analyzed by primer extension using 5′ -labeled primers to detect bottom-strand cDNAs (primer FB); the top-strand 5′-intron-integration junction (primer 5T); and top-strand DNAs (primer FT). Major products are diagrammed below the gel. (C) Requirements for bottom- and top-strand DNA synthesis. Reactions with the indicated components were incubated at 37°C for 30 min and then processed and analyzed in a denaturing 6% polyacrylamide gel, as described above. (D) Bottom- and top-strand products obtained with RNPs containing wild-type LtrA protein or an RT-deficient mutant LtrA (RT − ; YADD motif changed to YAAA). For simplicity, the bottom part of the gel with the labeled DNA substrate (S) is shown only for panel D. Asterisks indicate gels bands of the size expected for full-length bottom- and top-strand products.

Techniques Used: DNA Synthesis, Labeling, Sequencing, Incubation, Synthesized, Purification, Agarose Gel Electrophoresis, Mutagenesis

Assays of top- and bottom-strand DNA synthesis with extracts from  E. coli  mutants. DNA substrate labeled with  32 P at the 5′-end of the top (T) or bottom (B) strand were incubated with group II intron RNPs for 15 min at 37°C in reaction medium containing extracts from: (A) Keio deletion mutants and their parental wild-type strain BW25113. (B) Mutant strain C0719, which contains a  mariner -transposon at the site of a predicted sRNA, a  pnp  mutant, a  priB  deletion (non-Keio), and their parental wild-type strains; and (C) temperature-sensitive mutants and their parental wild-type strains. After phenol-CIA extraction and proteinase K digestion, samples were split into halves, which were incubated without or with RNase A+H at 37°C for 30 min. The products were analyzed in a denaturing 6% polyacrylamide gel, which was dried and scanned with a PhosphorImager. Extracts were confirmed to contain equal amounts of protein by SDS-polyacrylamide gels stained with Coomassie blue (not shown). The amount of radiolabel in the indicated product band or bands was normalized for the amount of substrate (S) in each lane and expressed as a percent of that in the parental wild-type strain (  Table 2 ). At least two assays were done for each mutant and were reproducible to within
Figure Legend Snippet: Assays of top- and bottom-strand DNA synthesis with extracts from E. coli mutants. DNA substrate labeled with 32 P at the 5′-end of the top (T) or bottom (B) strand were incubated with group II intron RNPs for 15 min at 37°C in reaction medium containing extracts from: (A) Keio deletion mutants and their parental wild-type strain BW25113. (B) Mutant strain C0719, which contains a mariner -transposon at the site of a predicted sRNA, a pnp mutant, a priB deletion (non-Keio), and their parental wild-type strains; and (C) temperature-sensitive mutants and their parental wild-type strains. After phenol-CIA extraction and proteinase K digestion, samples were split into halves, which were incubated without or with RNase A+H at 37°C for 30 min. The products were analyzed in a denaturing 6% polyacrylamide gel, which was dried and scanned with a PhosphorImager. Extracts were confirmed to contain equal amounts of protein by SDS-polyacrylamide gels stained with Coomassie blue (not shown). The amount of radiolabel in the indicated product band or bands was normalized for the amount of substrate (S) in each lane and expressed as a percent of that in the parental wild-type strain ( Table 2 ). At least two assays were done for each mutant and were reproducible to within

Techniques Used: DNA Synthesis, Labeling, Incubation, Mutagenesis, Staining

4) Product Images from "Modulation of dADAR-dependent RNA editing by the Drosophila fragile X mental retardation protein"

Article Title: Modulation of dADAR-dependent RNA editing by the Drosophila fragile X mental retardation protein

Journal: Nature Neuroscience

doi: 10.1038/nn.2950

dFMR1 biochemically interacts with dADAR in Drosophila S2 cell culture and in vivo (a) Structure of TAP (consisting of 2X FLAG and protein A sequences separated by a TEV cleavage site), dADAR(3A)-TAP and dADAR(3/4)-TAP constructs used to generate stable S2 cell lines. Constructs are under control of an inducible metallothionein (MT) promoter. ( b ) Western analysis showing expression of constructs in transfected S2 cells. Untransfected S2 cells were used as a negative control for the FLAG antibody. Astericks denote non-specific bands present in all samples that were detected by the anti-FLAG antibody. Molecular weight (MW) on left is measured in kilodaltons (kDa). ( c ) Eluates from TAP pulldown followed by TEV cleavage show that dFMR1 associates with dADAR-TAP in the presence of RNase A. Samples treated or untreated with RNase A are designated as (+) or (−), respectively. α-catenin was used as a loading control and does not associate with dADAR-TAP. A FLAG antibody was used to detect TAP constructs in input lanes. ( d ) RT-PCR analysis (upper panel) and ethidium bromide staining of total RNA (lower panel) on RNase A-treated and control lysates, showing efficient RNA degradation in RNase-treated samples. For RT-PCR analysis (upper panel), samples treated or untreated with RNase A are designated as (+) or (−), respectively. Primers against ribosomal protein 49 ( rp49 ) and α -Tubulin84D (α -Tub84D ) were used for PCR amplification. Molecular weight marker (MW) denotes size migration in basepairs (bp). For ethidium bromide staining of total RNA (lower panel), total RNA from TAP, dADAR(3A)-TAP, and dADAR(3/4)-TAP lysates were treated with DNase I (D), RNase A (R) or were untreated (C). ( e ) Co-IP experiments performed on head lysates prepared from w 1118 (control) and two independent endogenously tagged dADAR-HA fly lines, dAdar-HA 4.5. 2 and dAdar-HA 12.5.2 . An HA antibody was used to detect dADAR-HA and α-catenin was used as a loading control and negative control for the co-IP.
Figure Legend Snippet: dFMR1 biochemically interacts with dADAR in Drosophila S2 cell culture and in vivo (a) Structure of TAP (consisting of 2X FLAG and protein A sequences separated by a TEV cleavage site), dADAR(3A)-TAP and dADAR(3/4)-TAP constructs used to generate stable S2 cell lines. Constructs are under control of an inducible metallothionein (MT) promoter. ( b ) Western analysis showing expression of constructs in transfected S2 cells. Untransfected S2 cells were used as a negative control for the FLAG antibody. Astericks denote non-specific bands present in all samples that were detected by the anti-FLAG antibody. Molecular weight (MW) on left is measured in kilodaltons (kDa). ( c ) Eluates from TAP pulldown followed by TEV cleavage show that dFMR1 associates with dADAR-TAP in the presence of RNase A. Samples treated or untreated with RNase A are designated as (+) or (−), respectively. α-catenin was used as a loading control and does not associate with dADAR-TAP. A FLAG antibody was used to detect TAP constructs in input lanes. ( d ) RT-PCR analysis (upper panel) and ethidium bromide staining of total RNA (lower panel) on RNase A-treated and control lysates, showing efficient RNA degradation in RNase-treated samples. For RT-PCR analysis (upper panel), samples treated or untreated with RNase A are designated as (+) or (−), respectively. Primers against ribosomal protein 49 ( rp49 ) and α -Tubulin84D (α -Tub84D ) were used for PCR amplification. Molecular weight marker (MW) denotes size migration in basepairs (bp). For ethidium bromide staining of total RNA (lower panel), total RNA from TAP, dADAR(3A)-TAP, and dADAR(3/4)-TAP lysates were treated with DNase I (D), RNase A (R) or were untreated (C). ( e ) Co-IP experiments performed on head lysates prepared from w 1118 (control) and two independent endogenously tagged dADAR-HA fly lines, dAdar-HA 4.5. 2 and dAdar-HA 12.5.2 . An HA antibody was used to detect dADAR-HA and α-catenin was used as a loading control and negative control for the co-IP.

Techniques Used: Cell Culture, In Vivo, Construct, Western Blot, Expressing, Transfection, Negative Control, Molecular Weight, Reverse Transcription Polymerase Chain Reaction, Staining, Polymerase Chain Reaction, Amplification, Marker, Migration, Co-Immunoprecipitation Assay

5) Product Images from "Identification of Neuronal RNA Targets of TDP-43-containing Ribonucleoprotein Complexes *"

Article Title: Identification of Neuronal RNA Targets of TDP-43-containing Ribonucleoprotein Complexes *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.190884

Genomic distribution of reads from TDP-43 RNA library. A , Western blot ( IB ) of fractions from HeLa nuclear extracts (± RNase A) applied to a size exclusion column, blotted for TDP-43, hnRNPA1, and lamin A/C. Fraction 6 = blue dextran (2000 kDa), fraction 30 = apoferritin (443 kDa), fraction 40 = alcohol dehydrogenase (150 kDa), and fraction 54 = bovine serum albumin (54 kDa) (data not shown).  B , Western blot of fractions from rat brain nuclear extracts (± micrococcal nuclease (±  MNase )) blotted for TDP-43. Fraction 5 = blue dextran (2,000 kDa), fraction 45 = bovine serum albumin (54 kDa), and fraction 54 = carbonic anhydrase (29 kDa) (data not shown). Note that different size exclusion columns were used in  A  and  B. C ,  panel i , diagram of TDP-43 RIP method.  C ,  panel ii , representative Western blot of TDP-43 RIP.  IP:CTL , control immunoprecipitation.  D , distribution of raw reads from the TDP-43 library mapped to exonic and intronic genes regions.  CDS , coding sequence.  E , read density, number of reads per 1,000 mappable nucleotides per million reads ( mRPKM ) of gene regions from the TDP-43 library.
Figure Legend Snippet: Genomic distribution of reads from TDP-43 RNA library. A , Western blot ( IB ) of fractions from HeLa nuclear extracts (± RNase A) applied to a size exclusion column, blotted for TDP-43, hnRNPA1, and lamin A/C. Fraction 6 = blue dextran (2000 kDa), fraction 30 = apoferritin (443 kDa), fraction 40 = alcohol dehydrogenase (150 kDa), and fraction 54 = bovine serum albumin (54 kDa) (data not shown). B , Western blot of fractions from rat brain nuclear extracts (± micrococcal nuclease (± MNase )) blotted for TDP-43. Fraction 5 = blue dextran (2,000 kDa), fraction 45 = bovine serum albumin (54 kDa), and fraction 54 = carbonic anhydrase (29 kDa) (data not shown). Note that different size exclusion columns were used in A and B. C , panel i , diagram of TDP-43 RIP method. C , panel ii , representative Western blot of TDP-43 RIP. IP:CTL , control immunoprecipitation. D , distribution of raw reads from the TDP-43 library mapped to exonic and intronic genes regions. CDS , coding sequence. E , read density, number of reads per 1,000 mappable nucleotides per million reads ( mRPKM ) of gene regions from the TDP-43 library.

Techniques Used: Western Blot, CTL Assay, Immunoprecipitation, Sequencing

6) Product Images from "Copy number rather than epigenetic alterations are the major dictator of imprinted methylation in tumors"

Article Title: Copy number rather than epigenetic alterations are the major dictator of imprinted methylation in tumors

Journal: Nature Communications

doi: 10.1038/s41467-017-00639-9

Allelic expression analysis of KCNQ1OT1 in cancer cell lines. a Three cell lines with different copy-number status (COLO-824, copy number 2:1; HCC1954, copy number 4:2; MCF7 copy number 2:0) were assessed by Sanger sequencing of rs231359 polymorphism in the KCNQ1OT1 transcript in DNA and cDNA, respectively. The average methylation of the Kv DMR1 was determined from Infinium HM450k array data. b Representative RNA-FISH analysis of KCNQ1OT1 lncRNA-coated territory (green signal, white arrows) of individual nuclei with inserts representing zoomed in images of FISH signals. Nuclei were stained with DAPI. The quantification of KCNQ1OT1 expression signals represented as stacked bar charts indicates the percentage of nuclei displaying the indicated number of expression foci. c KCNQ1OT1 RNA-FISH performed on cells retreated with RNAse A
Figure Legend Snippet: Allelic expression analysis of KCNQ1OT1 in cancer cell lines. a Three cell lines with different copy-number status (COLO-824, copy number 2:1; HCC1954, copy number 4:2; MCF7 copy number 2:0) were assessed by Sanger sequencing of rs231359 polymorphism in the KCNQ1OT1 transcript in DNA and cDNA, respectively. The average methylation of the Kv DMR1 was determined from Infinium HM450k array data. b Representative RNA-FISH analysis of KCNQ1OT1 lncRNA-coated territory (green signal, white arrows) of individual nuclei with inserts representing zoomed in images of FISH signals. Nuclei were stained with DAPI. The quantification of KCNQ1OT1 expression signals represented as stacked bar charts indicates the percentage of nuclei displaying the indicated number of expression foci. c KCNQ1OT1 RNA-FISH performed on cells retreated with RNAse A

Techniques Used: Expressing, Sequencing, Methylation, Fluorescence In Situ Hybridization, Staining

7) Product Images from "A role for SOX9 in post-transcriptional processes: insights from the amphibian oocyte"

Article Title: A role for SOX9 in post-transcriptional processes: insights from the amphibian oocyte

Journal: Scientific Reports

doi: 10.1038/s41598-018-25356-1

Localization of SOX9 to the matrix of lateral loops is RNA-dependent. Nuclear spreads of X. laevis were immunostained for SOX9 [anti-Cter-SOX9 antibody (green, Alexa 488 IgG)] and SR proteins [mAb1H4 (red, Alexa 568 IgG)]. ( a – e ’) LBC from untreated nuclear spreads. ( f – j ’) LBC from nuclear spread digested with RNase A before immunostaining. ( a ’– e ’) Enlarged images of the regions marked by white boxes ( a–e ) show several lateral loops immunostained with SOX9 and SR antibodies. The arrowheads point to terminal granules. ( f ’– j ’) enlarged images of the regions marked by white boxes ( f–j ) show one lateral loop not stained with SOX9 and SR antibodies after digestion with Rnase A. The intensity of fluorescence in the different experiments was normalized with respect to their relevant controls (the secondary antibodies only). Wide field Leica microscopy. Scale bar: 10 μm.
Figure Legend Snippet: Localization of SOX9 to the matrix of lateral loops is RNA-dependent. Nuclear spreads of X. laevis were immunostained for SOX9 [anti-Cter-SOX9 antibody (green, Alexa 488 IgG)] and SR proteins [mAb1H4 (red, Alexa 568 IgG)]. ( a – e ’) LBC from untreated nuclear spreads. ( f – j ’) LBC from nuclear spread digested with RNase A before immunostaining. ( a ’– e ’) Enlarged images of the regions marked by white boxes ( a–e ) show several lateral loops immunostained with SOX9 and SR antibodies. The arrowheads point to terminal granules. ( f ’– j ’) enlarged images of the regions marked by white boxes ( f–j ) show one lateral loop not stained with SOX9 and SR antibodies after digestion with Rnase A. The intensity of fluorescence in the different experiments was normalized with respect to their relevant controls (the secondary antibodies only). Wide field Leica microscopy. Scale bar: 10 μm.

Techniques Used: Immunostaining, Staining, Fluorescence, Microscopy

8) Product Images from "Targeting and Anchoring Tudor in the Pole Plasm of the Drosophila Oocyte"

Article Title: Targeting and Anchoring Tudor in the Pole Plasm of the Drosophila Oocyte

Journal: PLoS ONE

doi: 10.1371/journal.pone.0014362

Relationship between Tud and Vas during oogenesis. (A-C) Distribution of GFP-Vas (green) and Tud (red) in wild-type (A) left, stage 6, and right, stage 8, (B) stage 9, and (C) stage 10 egg chambers. (A, upper panel) In stage 6 egg chambers Tud was enriched in the oocyte and in stage 8 transiently accumulated at the anterior margin of the oocyte, whereas GFP-Vas was predominantly detected in the nurse cells. (Lower panel) Both GFP-Vas and Tud decorated the membrane of nurse cell nuclei but the distribution of both proteins did not fully overlap; some Vas-containing particles were free of Tud. (B). In stage 9 egg chambers Tud and GFP-Vas began to accumulate at the posterior pole of the oocyte. (C, upper panel) Localization of GFP-Vas and Tud in nuage and pole plasm overlapped in a stage 10 egg chamber. (Lower panel) Higher magnification of the oocyte posterior cortex reveals the occurrence of particles containing both GFP-Vas and Tud in close proximity to the pole plasm (arrows). (D) RNA-dependent association of Tud with Vas. Immuno-detection of Tud in ovarian protein extracts separated by SDS-PAGE and blotted on PVDF membrane of wild-type females (first lane), homozygous tud 1 females (second lane), and affinity purified Vas-complexes isolated in presence (third lane) or absence of RNase inhibitors (fourth lane). Vas-complexes were purified from ovarian extracts representing ∼25-fold the amount of proteins loaded in the first lane. (E) RNase sensitive binding of Tud to Vas-complexes. Affinity purified Vas-complexes were isolated from ovarian protein extracts representing ∼75 fold the amount used in the first lane, as control, in presence of RNase inhibitors. Following purification the Vas-complexes were treated with RNase A and the Vas-complexes were isolated by centrifugation. Proteins in the pellet (P) and the supernatant (S) were separated and similarly analyzed for the occurrence of Tud as in D.
Figure Legend Snippet: Relationship between Tud and Vas during oogenesis. (A-C) Distribution of GFP-Vas (green) and Tud (red) in wild-type (A) left, stage 6, and right, stage 8, (B) stage 9, and (C) stage 10 egg chambers. (A, upper panel) In stage 6 egg chambers Tud was enriched in the oocyte and in stage 8 transiently accumulated at the anterior margin of the oocyte, whereas GFP-Vas was predominantly detected in the nurse cells. (Lower panel) Both GFP-Vas and Tud decorated the membrane of nurse cell nuclei but the distribution of both proteins did not fully overlap; some Vas-containing particles were free of Tud. (B). In stage 9 egg chambers Tud and GFP-Vas began to accumulate at the posterior pole of the oocyte. (C, upper panel) Localization of GFP-Vas and Tud in nuage and pole plasm overlapped in a stage 10 egg chamber. (Lower panel) Higher magnification of the oocyte posterior cortex reveals the occurrence of particles containing both GFP-Vas and Tud in close proximity to the pole plasm (arrows). (D) RNA-dependent association of Tud with Vas. Immuno-detection of Tud in ovarian protein extracts separated by SDS-PAGE and blotted on PVDF membrane of wild-type females (first lane), homozygous tud 1 females (second lane), and affinity purified Vas-complexes isolated in presence (third lane) or absence of RNase inhibitors (fourth lane). Vas-complexes were purified from ovarian extracts representing ∼25-fold the amount of proteins loaded in the first lane. (E) RNase sensitive binding of Tud to Vas-complexes. Affinity purified Vas-complexes were isolated from ovarian protein extracts representing ∼75 fold the amount used in the first lane, as control, in presence of RNase inhibitors. Following purification the Vas-complexes were treated with RNase A and the Vas-complexes were isolated by centrifugation. Proteins in the pellet (P) and the supernatant (S) were separated and similarly analyzed for the occurrence of Tud as in D.

Techniques Used: SDS Page, Affinity Purification, Isolation, Purification, Binding Assay, Centrifugation

9) Product Images from "Dependence of Intracellular and Exosomal microRNAs on Viral E6/E7 Oncogene Expression in HPV-positive Tumor Cells"

Article Title: Dependence of Intracellular and Exosomal microRNAs on Viral E6/E7 Oncogene Expression in HPV-positive Tumor Cells

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1004712

Characterization of exosomes secreted by HeLa cells used for small RNA deep sequencing. (A)  Immunoblot analysis of total cellular extract (30, 10 and 1 μg) from exosome-producing cells, and of 1 μg protein from exosome preparations. Hsc70, CD63, Annexin-1, CD9 and β-Actin: exosomal markers; EEA1: early endosome marker; GRP78: ER marker.  (B)  Visualization of exosomes by electron microscopy. Bar corresponds to 100 nm. (C) Characterization of cellular and exosomal RNA. Electropherograms of total RNA isolated from HeLa cells and from RNAse A-treated exosomes. Upper panel: total RNA contents; lower panel: small RNA contents. M = marker. Shown are representative images for siContr-1-treated samples.
Figure Legend Snippet: Characterization of exosomes secreted by HeLa cells used for small RNA deep sequencing. (A) Immunoblot analysis of total cellular extract (30, 10 and 1 μg) from exosome-producing cells, and of 1 μg protein from exosome preparations. Hsc70, CD63, Annexin-1, CD9 and β-Actin: exosomal markers; EEA1: early endosome marker; GRP78: ER marker. (B) Visualization of exosomes by electron microscopy. Bar corresponds to 100 nm. (C) Characterization of cellular and exosomal RNA. Electropherograms of total RNA isolated from HeLa cells and from RNAse A-treated exosomes. Upper panel: total RNA contents; lower panel: small RNA contents. M = marker. Shown are representative images for siContr-1-treated samples.

Techniques Used: Sequencing, Marker, Electron Microscopy, Isolation

10) Product Images from "Dependence of Intracellular and Exosomal microRNAs on Viral E6/E7 Oncogene Expression in HPV-positive Tumor Cells"

Article Title: Dependence of Intracellular and Exosomal microRNAs on Viral E6/E7 Oncogene Expression in HPV-positive Tumor Cells

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1004712

Characterization of exosomes secreted by HeLa cells used for small RNA deep sequencing. (A)  Immunoblot analysis of total cellular extract (30, 10 and 1 μg) from exosome-producing cells, and of 1 μg protein from exosome preparations. Hsc70, CD63, Annexin-1, CD9 and β-Actin: exosomal markers; EEA1: early endosome marker; GRP78: ER marker.  (B)  Visualization of exosomes by electron microscopy. Bar corresponds to 100 nm. (C) Characterization of cellular and exosomal RNA. Electropherograms of total RNA isolated from HeLa cells and from RNAse A-treated exosomes. Upper panel: total RNA contents; lower panel: small RNA contents. M = marker. Shown are representative images for siContr-1-treated samples.
Figure Legend Snippet: Characterization of exosomes secreted by HeLa cells used for small RNA deep sequencing. (A) Immunoblot analysis of total cellular extract (30, 10 and 1 μg) from exosome-producing cells, and of 1 μg protein from exosome preparations. Hsc70, CD63, Annexin-1, CD9 and β-Actin: exosomal markers; EEA1: early endosome marker; GRP78: ER marker. (B) Visualization of exosomes by electron microscopy. Bar corresponds to 100 nm. (C) Characterization of cellular and exosomal RNA. Electropherograms of total RNA isolated from HeLa cells and from RNAse A-treated exosomes. Upper panel: total RNA contents; lower panel: small RNA contents. M = marker. Shown are representative images for siContr-1-treated samples.

Techniques Used: Sequencing, Marker, Electron Microscopy, Isolation

11) Product Images from "HIV Tat controls RNA Polymerase II and the epigenetic landscape to transcriptionally reprogram target immune cells"

Article Title: HIV Tat controls RNA Polymerase II and the epigenetic landscape to transcriptionally reprogram target immune cells

Journal: eLife

doi: 10.7554/eLife.08955

The interaction between Tat and host cell chromatin appears to be primarily dictated by protein–protein interactions. ( A ) Fractionation scheme of Jurkat-GFP and Jurkat-Tat cells by increased salt extraction in the absence (–) or presence (+) of RNase A. Ch denotes the chromatin fraction. ( B ) Western blots of the samples prepared as in panel ( A ) with the indicated antibodies. The efficiency of RNase treatment was verified by electrophoresis of the purified RNA in an agarose gel stained with ethidium bromide. ( C ) ChIP assays to analyze the occupancy of GFP and Tat (FLAG) at the  CD69  promoter (-63 amplicon) in the absence (–) and presence (+) of RNase. ( D ) ChIP assays to analyze the occupancy of GFP and Tat (FLAG) at the  FAM46C  promoter (-182 amplicon) in the absence (–) and presence (+) of RNase. ( E ) ChIP assays to analyze the occupancy of GFP and Tat (FLAG) at the  CD1E  promoter (+4 amplicon) in the absence (–) and presence (+) of RNase. ( F ) ChIP assays to analyze the occupancy of GFP and Tat (FLAG) at the  EOMES  promoter (-57 amplicon) in the absence (–) and presence (+) of RNase (mean ± SEM; n = 3). ChIP, chromatin immunoprecipitation; GFP, green fluorescent protein; SEM, standard error of the mean. DOI: http://dx.doi.org/10.7554/eLife.08955.037
Figure Legend Snippet: The interaction between Tat and host cell chromatin appears to be primarily dictated by protein–protein interactions. ( A ) Fractionation scheme of Jurkat-GFP and Jurkat-Tat cells by increased salt extraction in the absence (–) or presence (+) of RNase A. Ch denotes the chromatin fraction. ( B ) Western blots of the samples prepared as in panel ( A ) with the indicated antibodies. The efficiency of RNase treatment was verified by electrophoresis of the purified RNA in an agarose gel stained with ethidium bromide. ( C ) ChIP assays to analyze the occupancy of GFP and Tat (FLAG) at the CD69 promoter (-63 amplicon) in the absence (–) and presence (+) of RNase. ( D ) ChIP assays to analyze the occupancy of GFP and Tat (FLAG) at the FAM46C promoter (-182 amplicon) in the absence (–) and presence (+) of RNase. ( E ) ChIP assays to analyze the occupancy of GFP and Tat (FLAG) at the CD1E promoter (+4 amplicon) in the absence (–) and presence (+) of RNase. ( F ) ChIP assays to analyze the occupancy of GFP and Tat (FLAG) at the EOMES promoter (-57 amplicon) in the absence (–) and presence (+) of RNase (mean ± SEM; n = 3). ChIP, chromatin immunoprecipitation; GFP, green fluorescent protein; SEM, standard error of the mean. DOI: http://dx.doi.org/10.7554/eLife.08955.037

Techniques Used: Fractionation, Western Blot, Electrophoresis, Purification, Agarose Gel Electrophoresis, Staining, Chromatin Immunoprecipitation, Amplification

12) Product Images from "HIV Tat controls RNA Polymerase II and the epigenetic landscape to transcriptionally reprogram target immune cells"

Article Title: HIV Tat controls RNA Polymerase II and the epigenetic landscape to transcriptionally reprogram target immune cells

Journal: eLife

doi: 10.7554/eLife.08955

The interaction between Tat and host cell chromatin appears to be primarily dictated by protein–protein interactions. ( A ) Fractionation scheme of Jurkat-GFP and Jurkat-Tat cells by increased salt extraction in the absence (–) or presence (+) of RNase A. Ch denotes the chromatin fraction. ( B ) Western blots of the samples prepared as in panel ( A ) with the indicated antibodies. The efficiency of RNase treatment was verified by electrophoresis of the purified RNA in an agarose gel stained with ethidium bromide. ( C ) ChIP assays to analyze the occupancy of GFP and Tat (FLAG) at the  CD69  promoter (-63 amplicon) in the absence (–) and presence (+) of RNase. ( D ) ChIP assays to analyze the occupancy of GFP and Tat (FLAG) at the  FAM46C  promoter (-182 amplicon) in the absence (–) and presence (+) of RNase. ( E ) ChIP assays to analyze the occupancy of GFP and Tat (FLAG) at the  CD1E  promoter (+4 amplicon) in the absence (–) and presence (+) of RNase. ( F ) ChIP assays to analyze the occupancy of GFP and Tat (FLAG) at the  EOMES  promoter (-57 amplicon) in the absence (–) and presence (+) of RNase (mean ± SEM; n = 3). ChIP, chromatin immunoprecipitation; GFP, green fluorescent protein; SEM, standard error of the mean. DOI: http://dx.doi.org/10.7554/eLife.08955.037
Figure Legend Snippet: The interaction between Tat and host cell chromatin appears to be primarily dictated by protein–protein interactions. ( A ) Fractionation scheme of Jurkat-GFP and Jurkat-Tat cells by increased salt extraction in the absence (–) or presence (+) of RNase A. Ch denotes the chromatin fraction. ( B ) Western blots of the samples prepared as in panel ( A ) with the indicated antibodies. The efficiency of RNase treatment was verified by electrophoresis of the purified RNA in an agarose gel stained with ethidium bromide. ( C ) ChIP assays to analyze the occupancy of GFP and Tat (FLAG) at the CD69 promoter (-63 amplicon) in the absence (–) and presence (+) of RNase. ( D ) ChIP assays to analyze the occupancy of GFP and Tat (FLAG) at the FAM46C promoter (-182 amplicon) in the absence (–) and presence (+) of RNase. ( E ) ChIP assays to analyze the occupancy of GFP and Tat (FLAG) at the CD1E promoter (+4 amplicon) in the absence (–) and presence (+) of RNase. ( F ) ChIP assays to analyze the occupancy of GFP and Tat (FLAG) at the EOMES promoter (-57 amplicon) in the absence (–) and presence (+) of RNase (mean ± SEM; n = 3). ChIP, chromatin immunoprecipitation; GFP, green fluorescent protein; SEM, standard error of the mean. DOI: http://dx.doi.org/10.7554/eLife.08955.037

Techniques Used: Fractionation, Western Blot, Electrophoresis, Purification, Agarose Gel Electrophoresis, Staining, Chromatin Immunoprecipitation, Amplification

13) Product Images from "Arf1p Provides an Unexpected Link between COPI Vesicles and mRNA in Saccharomyces cerevisiae D⃞"

Article Title: Arf1p Provides an Unexpected Link between COPI Vesicles and mRNA in Saccharomyces cerevisiae D⃞

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E04-05-0411

Arf1p and Pab1p are present in a ribonucleotide particle. (A and B) Pab1p and Arf1p coimmunoprecipitate. Pab1p and Arf1p were chromosomally appended with either a myc- or HA-tag. Yeast lysates were prepared from single- or double-tagged strains and subjected to immunoprecipitation with anti-myc or anti-HA antibodies. The precipitates were analyzed by immunoblot. Lanes 1 and 2 correspond to 1.7% of the lysate. (C) Pab1p–Arf1p interaction depends on mRNA. Yeast lysate from a wild-type strain was incubated with RNase A, DNase I, or mock treated. After the treatment an immunoprecipitation was performed with anti-Arf1p serum or a control serum and protein A-Sepharose. The precipitated proteins were detected by immunoblot. In lane 1, 1.7% of the lysate was loaded. (D) ASH1 mRNA is part of the Pab1p-Arf1p ribonucleotide particle even in the absence of the SHE machinery. A coimmunoprecipitation experiment was performed with affinity-purified anti-Arf1p antibodies. RNA was prepared from the precipitate and subjected to RT-PCR with primer specific for the indicated mRNAs. -RT indicates reactions in the absence of reverse transcriptase. In lanes 1 and 2, 1.7% of the lysate was loaded.
Figure Legend Snippet: Arf1p and Pab1p are present in a ribonucleotide particle. (A and B) Pab1p and Arf1p coimmunoprecipitate. Pab1p and Arf1p were chromosomally appended with either a myc- or HA-tag. Yeast lysates were prepared from single- or double-tagged strains and subjected to immunoprecipitation with anti-myc or anti-HA antibodies. The precipitates were analyzed by immunoblot. Lanes 1 and 2 correspond to 1.7% of the lysate. (C) Pab1p–Arf1p interaction depends on mRNA. Yeast lysate from a wild-type strain was incubated with RNase A, DNase I, or mock treated. After the treatment an immunoprecipitation was performed with anti-Arf1p serum or a control serum and protein A-Sepharose. The precipitated proteins were detected by immunoblot. In lane 1, 1.7% of the lysate was loaded. (D) ASH1 mRNA is part of the Pab1p-Arf1p ribonucleotide particle even in the absence of the SHE machinery. A coimmunoprecipitation experiment was performed with affinity-purified anti-Arf1p antibodies. RNA was prepared from the precipitate and subjected to RT-PCR with primer specific for the indicated mRNAs. -RT indicates reactions in the absence of reverse transcriptase. In lanes 1 and 2, 1.7% of the lysate was loaded.

Techniques Used: Immunoprecipitation, Incubation, Affinity Purification, Reverse Transcription Polymerase Chain Reaction

14) Product Images from "Ribonucleoprotein Particles Containing Non-Coding Y RNAs, Ro60, La and Nucleolin Are Not Required for Y RNA Function in DNA Replication"

Article Title: Ribonucleoprotein Particles Containing Non-Coding Y RNAs, Ro60, La and Nucleolin Are Not Required for Y RNA Function in DNA Replication

Journal: PLoS ONE

doi: 10.1371/journal.pone.0013673

Human Y RNAs are present in several distinct RNP complexes in cytosolic extract. The indicated proteins were immunoprecipitated (IP) from HeLa cytosolic extract and associated proteins and RNAs were analysed by Western blotting and qRT-PCR, respectively. (A) Protein analysis of Ro60 and La IPs. Apparent molecular weights of the precipitated protein bands are shown, the asterisk indicates the IgG heavy chain. As a reference, 10% of the input cell extract was loaded on the gel. Where indicated, extract was treated with RNase A prior to IP (RNase). (B) Protein analysis of nucleolin (NCL) IPs. (C) RNA content analysis of the Ro60, La and nucleolin (NCL) IPs. The relative amounts of all four hY RNAs and 5S rRNA in the indicated immunoprecipitates relative to mock immunoprecipitates were determined by qRT-PCR. Mean values of two independent experiments are shown.
Figure Legend Snippet: Human Y RNAs are present in several distinct RNP complexes in cytosolic extract. The indicated proteins were immunoprecipitated (IP) from HeLa cytosolic extract and associated proteins and RNAs were analysed by Western blotting and qRT-PCR, respectively. (A) Protein analysis of Ro60 and La IPs. Apparent molecular weights of the precipitated protein bands are shown, the asterisk indicates the IgG heavy chain. As a reference, 10% of the input cell extract was loaded on the gel. Where indicated, extract was treated with RNase A prior to IP (RNase). (B) Protein analysis of nucleolin (NCL) IPs. (C) RNA content analysis of the Ro60, La and nucleolin (NCL) IPs. The relative amounts of all four hY RNAs and 5S rRNA in the indicated immunoprecipitates relative to mock immunoprecipitates were determined by qRT-PCR. Mean values of two independent experiments are shown.

Techniques Used: Immunoprecipitation, Western Blot, Quantitative RT-PCR

15) Product Images from "The Lyme Disease Spirochete Borrelia burgdorferi Utilizes Multiple Ligands, Including RNA, for Interferon Regulatory Factor 3-Dependent Induction of Type I Interferon-Responsive Genes ▿"

Article Title: The Lyme Disease Spirochete Borrelia burgdorferi Utilizes Multiple Ligands, Including RNA, for Interferon Regulatory Factor 3-Dependent Induction of Type I Interferon-Responsive Genes ▿

Journal: Infection and Immunity

doi: 10.1128/IAI.01070-09

B. burgdorferi -derived RNA induces IFN profile gene transcription by BMDMs. C3H/HeJ BMDMs were treated for 6 h with 2 μg  B. burgdorferi  (Bb) RNA, genomic DNA, RNase A-digested  B. burgdorferi  RNA, DNase I-digested  B. burgdorferi  DNA, 0.7 μM CpG oligonucleotide, 7.4 × 10 6 /ml live  B. burgdorferi  organisms, live  B. burgdorferi  digested with DNase I or RNase A, 5 μg/ml sonicated  B. burgdorferi , or sonicate digested with DNase I or RNase A. RT-PCR transcripts are displayed as the number of copies of the gene of interest normalized to 1,000 copies of the mouse β-actin housekeeping gene. (A)  B. burgdorferi  RNA stimulates IFN-responsive gene transcription by BMDMs. Transcript levels for  Tyki and Cxcl10  are shown. Data are depicted as the means ± SEM and are representative of two independent experiments ( n  = 3). Statistical significance was assessed via the two-tailed, unpaired Student  t  test with Welch correction. *,  P
Figure Legend Snippet: B. burgdorferi -derived RNA induces IFN profile gene transcription by BMDMs. C3H/HeJ BMDMs were treated for 6 h with 2 μg B. burgdorferi (Bb) RNA, genomic DNA, RNase A-digested B. burgdorferi RNA, DNase I-digested B. burgdorferi DNA, 0.7 μM CpG oligonucleotide, 7.4 × 10 6 /ml live B. burgdorferi organisms, live B. burgdorferi digested with DNase I or RNase A, 5 μg/ml sonicated B. burgdorferi , or sonicate digested with DNase I or RNase A. RT-PCR transcripts are displayed as the number of copies of the gene of interest normalized to 1,000 copies of the mouse β-actin housekeeping gene. (A) B. burgdorferi RNA stimulates IFN-responsive gene transcription by BMDMs. Transcript levels for Tyki and Cxcl10 are shown. Data are depicted as the means ± SEM and are representative of two independent experiments ( n = 3). Statistical significance was assessed via the two-tailed, unpaired Student t test with Welch correction. *, P

Techniques Used: Derivative Assay, Sonication, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

16) Product Images from "HIPK2 and extrachromosomal histone H2B are separately recruited by Aurora-B for cytokinesis"

Article Title: HIPK2 and extrachromosomal histone H2B are separately recruited by Aurora-B for cytokinesis

Journal: Oncogene

doi: 10.1038/s41388-018-0191-6

H2B and HIPK2 localize at midbody independently of RNA.  a  Representative images of proliferating HCT116 DICER-proficient and -defective cells analyzed for midbody localization of histone H2B (green; left panels) and HIPK2 (green, right panels). Midbodies were marked with anti-β-tubulin Ab (β-Tub, red); DNA was visualized with Hoechst (blue).  b  Schematic representation of HeLa cells telophase enrichment with nocodazole, permeabilization, and treatment with RNase A, RNase III, and RNase H to cleave, respectively, single-strand RNA, double-strand RNA, and RNA/DNA hybrid. PBS and DNase I were used as negative controls.  c  After treatment, cells were fixed and stained with anti-β-tubulin Ab (red) and anti-H2B (green). DNA was marked with Hoechst (blue). Representative images of HeLa cells treated with the indicated enzymes. Each midbody visualized ( n >  50 in two independent experiments) was positive for H2B staining. Scale bar is 10 μm
Figure Legend Snippet: H2B and HIPK2 localize at midbody independently of RNA. a Representative images of proliferating HCT116 DICER-proficient and -defective cells analyzed for midbody localization of histone H2B (green; left panels) and HIPK2 (green, right panels). Midbodies were marked with anti-β-tubulin Ab (β-Tub, red); DNA was visualized with Hoechst (blue). b Schematic representation of HeLa cells telophase enrichment with nocodazole, permeabilization, and treatment with RNase A, RNase III, and RNase H to cleave, respectively, single-strand RNA, double-strand RNA, and RNA/DNA hybrid. PBS and DNase I were used as negative controls. c After treatment, cells were fixed and stained with anti-β-tubulin Ab (red) and anti-H2B (green). DNA was marked with Hoechst (blue). Representative images of HeLa cells treated with the indicated enzymes. Each midbody visualized ( n >  50 in two independent experiments) was positive for H2B staining. Scale bar is 10 μm

Techniques Used: Staining

17) Product Images from "High potency silencing by single-stranded boranophosphate siRNA"

Article Title: High potency silencing by single-stranded boranophosphate siRNA

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkl339

Nuclease stability of native and boranophosphate RNA. ss-ssRNAs were incubated with RNase A for the times indicated and then assessed for degradation by agarose gel electrophoresis. b, boranophosphate; n, native; A, adenosine; C, cytidine; U, uridine; 3, adenosine, cytidine and uridine.
Figure Legend Snippet: Nuclease stability of native and boranophosphate RNA. ss-ssRNAs were incubated with RNase A for the times indicated and then assessed for degradation by agarose gel electrophoresis. b, boranophosphate; n, native; A, adenosine; C, cytidine; U, uridine; 3, adenosine, cytidine and uridine.

Techniques Used: Incubation, Agarose Gel Electrophoresis

18) Product Images from "Stepwise RNP assembly at the site of H/ACA RNA transcription in human cells"

Article Title: Stepwise RNP assembly at the site of H/ACA RNA transcription in human cells

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200601105

Stable human U2OS cell lines expressing rat H/ACA RNA E3 from an inducible promoter. (A) Schematic of the construct that was stably integrated into the genome of the E3 and E3-minus cell lines. Expression was driven by a minimal CMV promoter (Pmin) under the control of the tetracycline response element (TRE) and was induced in the presence of doxycycline (dox) by the transactivator rtTA. The construct contained the polyadenylation/cleavage signal and transcriptional terminator (term) of the bovine growth hormone. The size of the probe used in the RNase protection assay (E) is indicated underneath. (B) Fluorescent micrographs of an E3 cell induced for transgene expression and assayed by FISH with Cy3-labeled probes to exon 1 and 2 (panel 1) and a Cy5-labeled probe to intron 1 (panel 2) and by direct fluorescence for MS2-GFP (panel 3) and for β-globin–CFP (panel 4). Panel 5 depicts the corresponding differential interference contrast image of the cell. (C and D) Triple fluorescence by FISH with probes to the induced rat E3 snoRNA (panel 1), which cross-hybridizes with the endogenous human E3 in uninduced cells (asterisks), and to exon 1 and 2 for identification of the transcription sites (panel 2), and by direct fluorescence of β-globin–CFP in peroxisomes (panel 3) to differentiate the induced cells from the uninduced cells. Insets (width = 2.6 μm) show a magnification of the transcription sites (arrows). (E) RNase protection assay. Autoradiograph of the sequencing gel used to separate the fragments protected from digestion by RNase A and T1. Yeast tRNA (lane 1) and total RNA from E3 (lanes 2 and 3) or E3-minus cells (lanes 4 and 5) isolated before (lanes 2 and 4) or 24 h after (lanes 3 and 5) induction with doxycycline were hybridized to the radiolabeled probe indicated in A. In addition, a separate probe corresponding to 100 nucleotides of SRP RNA was included in all samples as a control. The migrating positions of the protected fragments are indicated on the right. Note that because the probe corresponded to the rat E3 snoRNA of the integrated constructs, it protected only the smaller fragments of the endogenous human E3 snoRNA, which differed in 13 nucleotides from that of the rat.
Figure Legend Snippet: Stable human U2OS cell lines expressing rat H/ACA RNA E3 from an inducible promoter. (A) Schematic of the construct that was stably integrated into the genome of the E3 and E3-minus cell lines. Expression was driven by a minimal CMV promoter (Pmin) under the control of the tetracycline response element (TRE) and was induced in the presence of doxycycline (dox) by the transactivator rtTA. The construct contained the polyadenylation/cleavage signal and transcriptional terminator (term) of the bovine growth hormone. The size of the probe used in the RNase protection assay (E) is indicated underneath. (B) Fluorescent micrographs of an E3 cell induced for transgene expression and assayed by FISH with Cy3-labeled probes to exon 1 and 2 (panel 1) and a Cy5-labeled probe to intron 1 (panel 2) and by direct fluorescence for MS2-GFP (panel 3) and for β-globin–CFP (panel 4). Panel 5 depicts the corresponding differential interference contrast image of the cell. (C and D) Triple fluorescence by FISH with probes to the induced rat E3 snoRNA (panel 1), which cross-hybridizes with the endogenous human E3 in uninduced cells (asterisks), and to exon 1 and 2 for identification of the transcription sites (panel 2), and by direct fluorescence of β-globin–CFP in peroxisomes (panel 3) to differentiate the induced cells from the uninduced cells. Insets (width = 2.6 μm) show a magnification of the transcription sites (arrows). (E) RNase protection assay. Autoradiograph of the sequencing gel used to separate the fragments protected from digestion by RNase A and T1. Yeast tRNA (lane 1) and total RNA from E3 (lanes 2 and 3) or E3-minus cells (lanes 4 and 5) isolated before (lanes 2 and 4) or 24 h after (lanes 3 and 5) induction with doxycycline were hybridized to the radiolabeled probe indicated in A. In addition, a separate probe corresponding to 100 nucleotides of SRP RNA was included in all samples as a control. The migrating positions of the protected fragments are indicated on the right. Note that because the probe corresponded to the rat E3 snoRNA of the integrated constructs, it protected only the smaller fragments of the endogenous human E3 snoRNA, which differed in 13 nucleotides from that of the rat.

Techniques Used: Expressing, Construct, Stable Transfection, Rnase Protection Assay, Fluorescence In Situ Hybridization, Labeling, Fluorescence, Autoradiography, Sequencing, Isolation

19) Product Images from "Knockdown of BC200 RNA expression reduces cell migration and invasion by destabilizing mRNA for calcium-binding protein S100A11"

Article Title: Knockdown of BC200 RNA expression reduces cell migration and invasion by destabilizing mRNA for calcium-binding protein S100A11

Journal: RNA Biology

doi: 10.1080/15476286.2017.1297913

BC200 RNA-knockdown-associated changes in the ribosome footprint profile. (A) A schematic of the workflow used to profile ribosome footprints. First, ribosome bounded mRNAs were purified from HeLa cells transfected with siRNAs. RNase I degraded unbounded fractions of mRNAs. Next, ribosomes were purified by sucrose cushioning and mRNAs bounded by ribosomes were selectively extracted. Finally, the remained mRNA fragments were converted to sequencing libraries and sequencing was performed. (B) A volcano plot representing the fold change (x-axis, log 2  [Fold change]) and statistical significance (y-axis, −log 10   (C) GO of genes upregulated by knockdown of BC200 RNA. (D) GO of genes downregulated by knockdown of BC200 RNA.
Figure Legend Snippet: BC200 RNA-knockdown-associated changes in the ribosome footprint profile. (A) A schematic of the workflow used to profile ribosome footprints. First, ribosome bounded mRNAs were purified from HeLa cells transfected with siRNAs. RNase I degraded unbounded fractions of mRNAs. Next, ribosomes were purified by sucrose cushioning and mRNAs bounded by ribosomes were selectively extracted. Finally, the remained mRNA fragments were converted to sequencing libraries and sequencing was performed. (B) A volcano plot representing the fold change (x-axis, log 2 [Fold change]) and statistical significance (y-axis, −log 10 (C) GO of genes upregulated by knockdown of BC200 RNA. (D) GO of genes downregulated by knockdown of BC200 RNA.

Techniques Used: Purification, Transfection, Sequencing

20) Product Images from "Role of Mitochondrial Membrane Spherules in Flock House Virus Replication"

Article Title: Role of Mitochondrial Membrane Spherules in Flock House Virus Replication

Journal: Journal of Virology

doi: 10.1128/JVI.03080-15

Assignment of radiolabeled FHV RNA replication products. Total radiolabeled RNA products were resolved alongside radiolabeled in vitro -transcribed single-stranded FHV RNA1 and RNA 2 (A) or nuclease-digested FHV replication (Rep.) products (B). RNase A digests ssRNA, RNase H digests DNA:RNA hybrids, and micrococcal nuclease digests single-stranded and double-stranded RNA and DNA. The doublet for dsRNA1 was assigned based on resistance to RNase A digestion and increased size in comparison to ssRNA1 and was consistently observed throughout all experiments.
Figure Legend Snippet: Assignment of radiolabeled FHV RNA replication products. Total radiolabeled RNA products were resolved alongside radiolabeled in vitro -transcribed single-stranded FHV RNA1 and RNA 2 (A) or nuclease-digested FHV replication (Rep.) products (B). RNase A digests ssRNA, RNase H digests DNA:RNA hybrids, and micrococcal nuclease digests single-stranded and double-stranded RNA and DNA. The doublet for dsRNA1 was assigned based on resistance to RNase A digestion and increased size in comparison to ssRNA1 and was consistently observed throughout all experiments.

Techniques Used: In Vitro

21) Product Images from "Prp40 pre-mRNA processing factor 40 homolog B (PRPF40B) associates with SF1 and U2AF65 and modulates alternative pre-mRNA splicing in vivo"

Article Title: Prp40 pre-mRNA processing factor 40 homolog B (PRPF40B) associates with SF1 and U2AF65 and modulates alternative pre-mRNA splicing in vivo

Journal: RNA

doi: 10.1261/rna.047258.114

Colocalization of PRPF40B with nuclear speckles is not perturbed following RNase treatment or transcription inhibition. ( A ) After EGFP-PRPF40B expression, HEK293T cells were treated with RNase A. SRSF2 ( left  panel) and Sm ( right  panel) labeling were used
Figure Legend Snippet: Colocalization of PRPF40B with nuclear speckles is not perturbed following RNase treatment or transcription inhibition. ( A ) After EGFP-PRPF40B expression, HEK293T cells were treated with RNase A. SRSF2 ( left panel) and Sm ( right panel) labeling were used

Techniques Used: Inhibition, Expressing, Labeling

22) Product Images from "Modulation of HIV-like particle assembly in vitro by inositol phosphates"

Article Title: Modulation of HIV-like particle assembly in vitro by inositol phosphates

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

doi: 10.1073/pnas.191224698

Nuclease and salt resistance of particles assembled in vitro and of authentic, immature particles. Particles assembled in buffer B ( A ), buffer B plus 5% rabbit reticulocyte lysate ( B ), or buffer B plus 2 μM IP5 ( C ), were treated with RNase A (lanes 2) or NaCl (lanes 3). Gag proteins in particles pelleted after treatment were examined by SDS/PAGE and Coomassie blue staining. We estimate that > 90% of the Gag protein was solubilized in A (lanes 2 and 3), whereas > 90% remained pelletable in B and C (lanes 2 and 3). ( D ) Immature MoMuLV and HIV-1 virions produced in mammalian cells were analyzed separately (lanes 1 and 2, respectively) or were mixed together (lanes 3–9) and treated with RNase A (lanes 6 and 7) or NaCl (lanes 8 and 9). They were also digested with HIV-1 PR to confirm that the lipid envelope was removed by the detergent (lane 3), or sedimented without RNase or NaCl treatment to confirm that the particles are stable in buffer B (lanes 4 and 5). Gag proteins in the samples in D were detected by immunoblotting with antibodies against the CA proteins of both HIV-1 and MoMuLV. We estimate that > 90% of the HIV-1 Gag protein was still pelletable after NaCl or RNase treatment. P, pellet; S, supernatant.
Figure Legend Snippet: Nuclease and salt resistance of particles assembled in vitro and of authentic, immature particles. Particles assembled in buffer B ( A ), buffer B plus 5% rabbit reticulocyte lysate ( B ), or buffer B plus 2 μM IP5 ( C ), were treated with RNase A (lanes 2) or NaCl (lanes 3). Gag proteins in particles pelleted after treatment were examined by SDS/PAGE and Coomassie blue staining. We estimate that > 90% of the Gag protein was solubilized in A (lanes 2 and 3), whereas > 90% remained pelletable in B and C (lanes 2 and 3). ( D ) Immature MoMuLV and HIV-1 virions produced in mammalian cells were analyzed separately (lanes 1 and 2, respectively) or were mixed together (lanes 3–9) and treated with RNase A (lanes 6 and 7) or NaCl (lanes 8 and 9). They were also digested with HIV-1 PR to confirm that the lipid envelope was removed by the detergent (lane 3), or sedimented without RNase or NaCl treatment to confirm that the particles are stable in buffer B (lanes 4 and 5). Gag proteins in the samples in D were detected by immunoblotting with antibodies against the CA proteins of both HIV-1 and MoMuLV. We estimate that > 90% of the HIV-1 Gag protein was still pelletable after NaCl or RNase treatment. P, pellet; S, supernatant.

Techniques Used: In Vitro, SDS Page, Staining, Produced

23) Product Images from "Raver1, a dual compartment protein, is a ligand for PTB/hnRNPI and microfilament attachment proteins"

Article Title: Raver1, a dual compartment protein, is a ligand for PTB/hnRNPI and microfilament attachment proteins

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200105044

Raver1 interacts with the PTB/hnRNPI protein. (A) Colocalization of PTB/hnRNPI and raver1 in C2C12 myoblasts as seen by double immunofluorescence with the respective monoclonal antibodies. Both proteins are localized in the nucleus and highly concentrated in the perinucleolar bodies (arrowheads). (B and C) Coprecipitation of endogenous raver1 with PTB/hnRNPI from nontransfected C2C12 myoblasts (B) and HeLa cells transfected with FLAG-equipped raver1 or FLAG-ZBP1 (C), and analysis of the precipitates by SDS-PAGE and immunoblotting. Antibodies used for immunoprecipitation (against PTB/hnRNPI, raver1, ZBP1, and FLAG) are indicated on the top; those used for immunoblots are shown on the right. Cell extracts were treated with RNAsin or with RNAse A to either protect or remove putatively bound RNA. Note that PTB/hnRNPI and raver1 coprecipitate independently of RNA, whereas ZBP1 does not form complexes with raver1 under either experimental condition. CE, immunoblots of the total extracts, to demonstrate the expression of the relevant proteins. (D) Liquid β-galactosidase assay of the yeast two-hybrid system to identify the raver1 domain interacting with murine PTB/hnRNPI. β-Galactosidase activity was found highest with intact raver1, and some activity remained with the COOH-terminal deletion fragment, whereas the NH 2 -terminal deletion fragment was inactive. Means and calculated standard deviation of three independent experiments are indicated. Bars, 10 μm.
Figure Legend Snippet: Raver1 interacts with the PTB/hnRNPI protein. (A) Colocalization of PTB/hnRNPI and raver1 in C2C12 myoblasts as seen by double immunofluorescence with the respective monoclonal antibodies. Both proteins are localized in the nucleus and highly concentrated in the perinucleolar bodies (arrowheads). (B and C) Coprecipitation of endogenous raver1 with PTB/hnRNPI from nontransfected C2C12 myoblasts (B) and HeLa cells transfected with FLAG-equipped raver1 or FLAG-ZBP1 (C), and analysis of the precipitates by SDS-PAGE and immunoblotting. Antibodies used for immunoprecipitation (against PTB/hnRNPI, raver1, ZBP1, and FLAG) are indicated on the top; those used for immunoblots are shown on the right. Cell extracts were treated with RNAsin or with RNAse A to either protect or remove putatively bound RNA. Note that PTB/hnRNPI and raver1 coprecipitate independently of RNA, whereas ZBP1 does not form complexes with raver1 under either experimental condition. CE, immunoblots of the total extracts, to demonstrate the expression of the relevant proteins. (D) Liquid β-galactosidase assay of the yeast two-hybrid system to identify the raver1 domain interacting with murine PTB/hnRNPI. β-Galactosidase activity was found highest with intact raver1, and some activity remained with the COOH-terminal deletion fragment, whereas the NH 2 -terminal deletion fragment was inactive. Means and calculated standard deviation of three independent experiments are indicated. Bars, 10 μm.

Techniques Used: Immunofluorescence, Transfection, SDS Page, Immunoprecipitation, Western Blot, Expressing, Activity Assay, Standard Deviation

24) Product Images from "Visualizing protein-RNA interactions inside cells by fluorescence resonance energy transfer"

Article Title: Visualizing protein-RNA interactions inside cells by fluorescence resonance energy transfer

Journal: RNA

doi: 10.1261/rna.1307809

Staining nucleic acids with SytoxOrange can be used as FRET acceptor for YFP-tagged binding proteins. ( A , B ) Quantification of the RNase and DNase digest. HeLa cells stained with SytoxOrange, after RNase A or DNase I treatment are shown with identical
Figure Legend Snippet: Staining nucleic acids with SytoxOrange can be used as FRET acceptor for YFP-tagged binding proteins. ( A , B ) Quantification of the RNase and DNase digest. HeLa cells stained with SytoxOrange, after RNase A or DNase I treatment are shown with identical

Techniques Used: Staining, Binding Assay

25) Product Images from "Mesenchymal stem cell secretes microparticles enriched in pre-microRNAs"

Article Title: Mesenchymal stem cell secretes microparticles enriched in pre-microRNAs

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkp857

Secreted RNAs were sequestered in phospholipid vesicles. ( A ) Untreated CM or CM pretreated with SDS-based lysis buffer, cyclodextrin or phospholipase A2 was incubated with RNase A. After incubation, the CMs were extracted for RNA and the RNAs were resolved on a 15% TBE–urea gel. ( B ) RNA was extracted from CM without RNase inhibitor (lane 2), with RNase inhibitor (lane 3) or CM pretreated with cyclodextrin (lane 4), SDS-based lysis buffer (lane 5) or phospholipase A2 (lane 6).
Figure Legend Snippet: Secreted RNAs were sequestered in phospholipid vesicles. ( A ) Untreated CM or CM pretreated with SDS-based lysis buffer, cyclodextrin or phospholipase A2 was incubated with RNase A. After incubation, the CMs were extracted for RNA and the RNAs were resolved on a 15% TBE–urea gel. ( B ) RNA was extracted from CM without RNase inhibitor (lane 2), with RNase inhibitor (lane 3) or CM pretreated with cyclodextrin (lane 4), SDS-based lysis buffer (lane 5) or phospholipase A2 (lane 6).

Techniques Used: Lysis, Incubation

26) Product Images from "RNA-mediated dimerization of the human deoxycytidine deaminase APOBEC3H influences enzyme activity and interaction with nucleic acids"

Article Title: RNA-mediated dimerization of the human deoxycytidine deaminase APOBEC3H influences enzyme activity and interaction with nucleic acids

Journal: Journal of molecular biology

doi: 10.1016/j.jmb.2018.11.006

Deamination of ssDNA by GST-A3H. Deamination activity was tested on an 85 nt ssDNA with two 5′CTC deamination motifs separated by 30 nt. (A-B) A3H WT can deaminate ssDNA in the presence and absence of bound cellular RNA. (C-D) Monomeric A3H mutants have disrupted ssDNA deaminase activity. Using different MgCl 2  concentrations that were found to enhance GST-A3H WT activity, the deamination activity of A3H mutants were tested in the presence of RNase A. (C-D) The A3H GST-Y112A/, GST-W115A and GST-R175E/R176E were not active on ssDNA. A representative image is shown from three independent experiments. The S.D. was calculated from three independent experiments and is shown below the gel or for panel (B) is represented by error bars. Some error bars in (B) are obscured by the symbol.
Figure Legend Snippet: Deamination of ssDNA by GST-A3H. Deamination activity was tested on an 85 nt ssDNA with two 5′CTC deamination motifs separated by 30 nt. (A-B) A3H WT can deaminate ssDNA in the presence and absence of bound cellular RNA. (C-D) Monomeric A3H mutants have disrupted ssDNA deaminase activity. Using different MgCl 2 concentrations that were found to enhance GST-A3H WT activity, the deamination activity of A3H mutants were tested in the presence of RNase A. (C-D) The A3H GST-Y112A/, GST-W115A and GST-R175E/R176E were not active on ssDNA. A representative image is shown from three independent experiments. The S.D. was calculated from three independent experiments and is shown below the gel or for panel (B) is represented by error bars. Some error bars in (B) are obscured by the symbol.

Techniques Used: Activity Assay

27) Product Images from "Sequence Variability of Borna Disease Virus: Resistance to Superinfection May Contribute to High Genome Stability in Persistently Infected Cells"

Article Title: Sequence Variability of Borna Disease Virus: Resistance to Superinfection May Contribute to High Genome Stability in Persistently Infected Cells

Journal: Journal of Virology

doi:

RPA for the detection of sequence variations between different strains of BDV. Nucleotide differences between the BDV strains He/80 and V were visualized by RPA using a 535-nt RNA probe (lane 1) complementary to the M and G open reading frames of strain He/80. Analysis was carried out using 10 μg of tRNA (lane 2), RNA samples (10 μg) prepared from total cell lysates of uninfected (uninf.) C6 cells (lane 3), C6 cells infected with BDV strain He/80 (lanes 4 and 5), or C6 cells infected with BDV strain V (lanes 6 and 7). Low (0.02-μg) or high (4-μg) concentrations of RNase A were used (+) as indicated. The repertoire of RNA species and the expected gel positions of the corresponding RPA signals are indicated on the right. The arrows mark signals resulting from RNA species with slightly different sequences. The numbers to the left indicate the mobility of molecular size markers (in nucleotides).
Figure Legend Snippet: RPA for the detection of sequence variations between different strains of BDV. Nucleotide differences between the BDV strains He/80 and V were visualized by RPA using a 535-nt RNA probe (lane 1) complementary to the M and G open reading frames of strain He/80. Analysis was carried out using 10 μg of tRNA (lane 2), RNA samples (10 μg) prepared from total cell lysates of uninfected (uninf.) C6 cells (lane 3), C6 cells infected with BDV strain He/80 (lanes 4 and 5), or C6 cells infected with BDV strain V (lanes 6 and 7). Low (0.02-μg) or high (4-μg) concentrations of RNase A were used (+) as indicated. The repertoire of RNA species and the expected gel positions of the corresponding RPA signals are indicated on the right. The arrows mark signals resulting from RNA species with slightly different sequences. The numbers to the left indicate the mobility of molecular size markers (in nucleotides).

Techniques Used: Recombinase Polymerase Amplification, Sequencing, Infection

Related Articles

Clone Assay:

Article Title: HIV Tat controls RNA Polymerase II and the epigenetic landscape to transcriptionally reprogram target immune cells
Article Snippet: DNA samples were then treated with 5 μl of RNase A (Roche) for 30 min at 37°C, and then mixed with 7 ml of phenol–chloroform vigorously and centrifuged for 15 min at 2200 g at room temperature. .. Random ligation matrix was prepared by digestion with restriction enzyme and ligation with BAC clones containing the respective genes examined from the Children’s Hospital Oakland Research Institute (CHORI, Oakland, CA).

Centrifugation:

Article Title: HIV Tat controls RNA Polymerase II and the epigenetic landscape to transcriptionally reprogram target immune cells
Article Snippet: Nuclei were pelleted by centrifugation (5 min spin at 2,000 g at 4°C), the supernatant saved as the cytoplasmic extract (∼0.5–0.6 ml), and the nuclear pellet was washed twice, with 1 ml buffer A. .. The pellet was similarly resuspended in 2× pellet volume of buffer B containing 600 mM NaCl, vortexed for 10 s, treated with RNAse A (Roche) or BSA (mock treatment) at a concentration of 10 μg/ml for 60 min at 4°C and then spun at 6000 g for 5 min.

Article Title: Ternatin and improved synthetic variants kill cancer cells by targeting the elongation factor-1A ternary complex
Article Snippet: Crude lysates were clarified by centrifugation (15 min at 14,000 rpm), protein was quantified by Bradford assay, and the protein concentration was brought to 1.5 mg/mL with lysis buffer. .. For samples treated with RNase A, RNase A (Roche) was added to lysates (10 mg/mL final concentration) and incubated for 20 min at RT.

Article Title: Evidence that Lin28 stimulates translation by recruiting RNA helicase A to polysomes
Article Snippet: .. Cell pellet was resuspended in 400 µl of gentle lysis buffer [10 mM Tris–HCl at pH 7.5, 10 mM NaCl, 10 mM EDTA, 0.5% Triton X-100, 1 mM PMSF, 1× protease inhibitor cocktail (Calbiochem), 1 mM DTT and 10 µg/ml of RNase A (Roche)] and incubated on ice for 15 min. Insoluble materials were removed by centrifugation at 13 400 g in a microcentrifuge at 4°C for 15 min. NaCl was added to the cleared lysate to a final concentration of 200 mM, and 350 µl of the lysate incubated with 20 µl of protein-A sepharose beads pre-bound with 10 µl of anti-Lin28 antibody, pre-immune IgG ( C), or 10 µg of anti-Flag M2 antibody ( B) at 4°C overnight. .. The next day, beads were washed and bound fractions eluted by 3× SDS-sample buffer by heating at 95°C for 5 min. Proteins were resolved by SDS–PAGE, followed by western blot analysis.

Article Title: Targeting and Anchoring Tudor in the Pole Plasm of the Drosophila Oocyte
Article Snippet: The protein-A beads were separated at low speed centrifugation, suspended in IP buffer, and washed 5 times. .. For the release experiment the immuno-complexes were washed extensively and incubated for 20 minutes at room temperature with 50μg of RNase A (Roche).

DNA Ligation:

Article Title: HIV Tat controls RNA Polymerase II and the epigenetic landscape to transcriptionally reprogram target immune cells
Article Snippet: DNA ligation was performed by incubation with 2000 units of T4 DNA ligase (New England Biolabs) for 4 hr at 16°C followed by 30 min at room temperature. .. DNA samples were then treated with 5 μl of RNase A (Roche) for 30 min at 37°C, and then mixed with 7 ml of phenol–chloroform vigorously and centrifuged for 15 min at 2200 g at room temperature.

Cell Cycle Assay:

Article Title: Dependence of Intracellular and Exosomal microRNAs on Viral E6/E7 Oncogene Expression in HPV-positive Tumor Cells
Article Snippet: Cell cycle analyses For cell cycle analysis, cells were trypsinized 72 h after transfection, washed in ice-cold PBS and fixed in 80% cold ethanol overnight at -20°C. .. Subsequently cells were pelleted, resuspended in phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2 HPO4 , 1.4 mM KH2 PO4 , pH 7.4) containing 1 mg/ml RNase A (Roche Diagnostics) and 25 μg/ml propidium iodide (Sigma-Aldrich) and incubated for 30 min at room temperature (RT).

Cell Fractionation:

Article Title: HIV Tat controls RNA Polymerase II and the epigenetic landscape to transcriptionally reprogram target immune cells
Article Snippet: Paragraph title: Cell fractionation by sequential salt extraction ... The pellet was similarly resuspended in 2× pellet volume of buffer B containing 600 mM NaCl, vortexed for 10 s, treated with RNAse A (Roche) or BSA (mock treatment) at a concentration of 10 μg/ml for 60 min at 4°C and then spun at 6000 g for 5 min.

Cytometry:

Article Title: Dependence of Intracellular and Exosomal microRNAs on Viral E6/E7 Oncogene Expression in HPV-positive Tumor Cells
Article Snippet: Subsequently cells were pelleted, resuspended in phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2 HPO4 , 1.4 mM KH2 PO4 , pH 7.4) containing 1 mg/ml RNase A (Roche Diagnostics) and 25 μg/ml propidium iodide (Sigma-Aldrich) and incubated for 30 min at room temperature (RT). .. Cell cycle analyses were performed by fluorescence-activated cell sorting (FACS) using a FACSCalibur Flow Cytometer (BD Biosciences, Heidelberg, Germany) with CellQuest Pro software provided by the manufacturer.

Immunostaining:

Article Title: A role for SOX9 in post-transcriptional processes: insights from the amphibian oocyte
Article Snippet: They were then digested 30 min at 37° with RNase A (Roche Life Science, France) (20μg/ml). .. After washing 3 × 10 min in PBS, preparations were processed for immunostaining experiments as described below.

Electrophoresis:

Article Title: Determinants of R-loop formation at convergent bidirectionally transcribed trinucleotide repeats
Article Snippet: .. RNase treatment and electrophoresis To analyze hybrid formation, samples from in vitro transcription reactions were divided into three (5 μl each) and treated with either TE (transcription control), 1 μg RNase A (Roche) or 10U of RNase T1 (Roche), 1 μg RNase A + 1 U RNase H (Roche) or 10U RNase T1 + 1 U RNase H as stated in a final volume of 10 μl for 20 min at room temperature. .. All in vitro transcription reaction products were analyzed on 0.8% agarose gels run in 1× Tris–Borate–EDTA buffer at 80 V for 5 h. Gels were subsequently stained with ethidium bromide (0.5 μg/ml) to allow visualization of the nucleic acid products under UV light.

Quantitation Assay:

Article Title: Dependence of Intracellular and Exosomal microRNAs on Viral E6/E7 Oncogene Expression in HPV-positive Tumor Cells
Article Snippet: Subsequently cells were pelleted, resuspended in phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2 HPO4 , 1.4 mM KH2 PO4 , pH 7.4) containing 1 mg/ml RNase A (Roche Diagnostics) and 25 μg/ml propidium iodide (Sigma-Aldrich) and incubated for 30 min at room temperature (RT). .. Quantitation of the percentage of cells in the individual phases was performed using FlowJo software (Tree Star, Ashland, OR, USA), applying the Dean-Jett-Fox model [ ].

Activity Assay:

Article Title: Genetic and Biochemical Assays Reveal a Key Role for Replication Restart Proteins in Group II Intron Retrohoming
Article Snippet: Assays were carried out in 20 µl of reaction mixture containing 50 nM DNA substrate, 3 µl of in vitro reconstituted Ll.LtrB RNPs (5–10 µg based on O.D.260 ; RNPs prepared as described in ref. ), 6 µl of S12 extract, 20 µM carrier DNA oligonucleotide ( 5′-GTGATGTCTGAAAAGAACGGGAAG ) as protection against DNase activity, 56.4 mM Tris-acetate buffer, pH 7.5, 100 mM potassium acetate, 35.9 mM ammonium acetate, 24 mM magnesium acetate, 1.5 mM ATP, 1 mM e ach of dATP, dCTP, dGTP, and dTTP (collectively denoted dNTPs), 500 µM of CTP, GTP, and UTP, 5 mM phosphoenolpyruvate, 50 µg/ml pyruvate kinase, 2 units/µl RNaseOUT (Invitrogen), and 1% (v/v) protease inhibitor cocktail [made by dissolving a mini EDTA-free tablet (Roche) in 1 ml of RNase-free water]. .. For RNase treatment, 0.4 units RNase H (Invitrogen) and 0.1 µg RNase A (Roche) were added, and the sample was incubated for 30 min at 37°C before the ethanol-precipitation step.

Expressing:

Article Title: Copy number rather than epigenetic alterations are the major dictator of imprinted methylation in tumors
Article Snippet: RNA FISH Expression of the lncRNA KCNQ1OT1 assessed by RNA FISH. .. Control slides were treated with RNAse A (Roche) treatment for 1 h at 37 °C and subsequently washed twice in 2× SSC.

Article Title: Modulation of dADAR-dependent RNA editing by the Drosophila fragile X mental retardation protein
Article Snippet: Tandem affinity purification (TAP) assay for dADAR-TAP-expressing cells For dADAR-TAP pulldown assays, Drosophila S2 cells expressing pCoBlast-dADAR-3A-TAP, pCoBlast-dADAR-3/4-TAP, or pCoBlast-TAP constructs were used for the TAP assay. .. TAP purification was performed as previously described with the following modifications: After overnight incubation with IgG sepharose, beads were washed in TEV cleavage buffer five times, with the fourth wash being supplemented with RNase A (Roche, final concentration 0.05 μg/μl) for 1 hr.

Bradford Assay:

Article Title: Ternatin and improved synthetic variants kill cancer cells by targeting the elongation factor-1A ternary complex
Article Snippet: Crude lysates were clarified by centrifugation (15 min at 14,000 rpm), protein was quantified by Bradford assay, and the protein concentration was brought to 1.5 mg/mL with lysis buffer. .. For samples treated with RNase A, RNase A (Roche) was added to lysates (10 mg/mL final concentration) and incubated for 20 min at RT.

Western Blot:

Article Title: Evidence that Lin28 stimulates translation by recruiting RNA helicase A to polysomes
Article Snippet: Cell pellet was resuspended in 400 µl of gentle lysis buffer [10 mM Tris–HCl at pH 7.5, 10 mM NaCl, 10 mM EDTA, 0.5% Triton X-100, 1 mM PMSF, 1× protease inhibitor cocktail (Calbiochem), 1 mM DTT and 10 µg/ml of RNase A (Roche)] and incubated on ice for 15 min. Insoluble materials were removed by centrifugation at 13 400 g in a microcentrifuge at 4°C for 15 min. NaCl was added to the cleared lysate to a final concentration of 200 mM, and 350 µl of the lysate incubated with 20 µl of protein-A sepharose beads pre-bound with 10 µl of anti-Lin28 antibody, pre-immune IgG ( C), or 10 µg of anti-Flag M2 antibody ( B) at 4°C overnight. .. The next day, beads were washed and bound fractions eluted by 3× SDS-sample buffer by heating at 95°C for 5 min. Proteins were resolved by SDS–PAGE, followed by western blot analysis.

Article Title: Targeting and Anchoring Tudor in the Pole Plasm of the Drosophila Oocyte
Article Snippet: Proteins associated with the beads were then analyzed on a western blot for the presence of Tud by using an enhanced chemiluminiscence system. .. For the release experiment the immuno-complexes were washed extensively and incubated for 20 minutes at room temperature with 50μg of RNase A (Roche).

Article Title: Modulation of dADAR-dependent RNA editing by the Drosophila fragile X mental retardation protein
Article Snippet: TAP purification was performed as previously described with the following modifications: After overnight incubation with IgG sepharose, beads were washed in TEV cleavage buffer five times, with the fourth wash being supplemented with RNase A (Roche, final concentration 0.05 μg/μl) for 1 hr. .. TEV cleavage was performed for 4 hrs and complexes were collected and run on SDS-PAGE followed by Western analysis.

Hybridization:

Article Title: Copy number rather than epigenetic alterations are the major dictator of imprinted methylation in tumors
Article Snippet: Control slides were treated with RNAse A (Roche) treatment for 1 h at 37 °C and subsequently washed twice in 2× SSC. .. A pre-hybridization step involving incubating the fixed slides in 15% formamide/2× SSC for 20 min at room temperature was performed before hybridization.

Transfection:

Article Title: Dependence of Intracellular and Exosomal microRNAs on Viral E6/E7 Oncogene Expression in HPV-positive Tumor Cells
Article Snippet: Cell cycle analyses For cell cycle analysis, cells were trypsinized 72 h after transfection, washed in ice-cold PBS and fixed in 80% cold ethanol overnight at -20°C. .. Subsequently cells were pelleted, resuspended in phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2 HPO4 , 1.4 mM KH2 PO4 , pH 7.4) containing 1 mg/ml RNase A (Roche Diagnostics) and 25 μg/ml propidium iodide (Sigma-Aldrich) and incubated for 30 min at room temperature (RT).

Article Title: Evidence that Lin28 stimulates translation by recruiting RNA helicase A to polysomes
Article Snippet: Co-immunoprecipitation To examine the interactions between Lin28 and RHA, 8 × 106 HEK293 (or PA-1) cells were transfected with 2 µg of Flag-Lin28 (Flag-28ΔC, Flag-28ΔN or empty vector), with or without co-transfection of 6 µg of Flag-N300 in a 6 cm plate scale (total DNA per plate was 8 µg). .. Cell pellet was resuspended in 400 µl of gentle lysis buffer [10 mM Tris–HCl at pH 7.5, 10 mM NaCl, 10 mM EDTA, 0.5% Triton X-100, 1 mM PMSF, 1× protease inhibitor cocktail (Calbiochem), 1 mM DTT and 10 µg/ml of RNase A (Roche)] and incubated on ice for 15 min. Insoluble materials were removed by centrifugation at 13 400 g in a microcentrifuge at 4°C for 15 min. NaCl was added to the cleared lysate to a final concentration of 200 mM, and 350 µl of the lysate incubated with 20 µl of protein-A sepharose beads pre-bound with 10 µl of anti-Lin28 antibody, pre-immune IgG ( C), or 10 µg of anti-Flag M2 antibody ( B) at 4°C overnight.

Ligation:

Article Title: HIV Tat controls RNA Polymerase II and the epigenetic landscape to transcriptionally reprogram target immune cells
Article Snippet: Digested nuclei were transferred to 50 ml Falcon tube and incubated with 6.125 ml of 1.15 X ligation buffer and 375 µl of 20% (v/v) Triton X-100 for 1 hr at 37°C with constant shaking. .. DNA samples were then treated with 5 μl of RNase A (Roche) for 30 min at 37°C, and then mixed with 7 ml of phenol–chloroform vigorously and centrifuged for 15 min at 2200 g at room temperature.

Protease Inhibitor:

Article Title: HIV Tat controls RNA Polymerase II and the epigenetic landscape to transcriptionally reprogram target immune cells
Article Snippet: Cell fractionation by sequential salt extraction 1 × 108 cells (Jurkat-GFP and -Tat) were collected from 100 ml of culture media (grown at 1 × 106 cells/ml) and washed with 2 ml of cold 1× phosphate buffered saline (PBS) plus ethylendiaminetetraacetic acid (EDTA)-free protease inhibitor (PI) cocktail (Roche). .. The pellet was similarly resuspended in 2× pellet volume of buffer B containing 600 mM NaCl, vortexed for 10 s, treated with RNAse A (Roche) or BSA (mock treatment) at a concentration of 10 μg/ml for 60 min at 4°C and then spun at 6000 g for 5 min.

Article Title: Ternatin and improved synthetic variants kill cancer cells by targeting the elongation factor-1A ternary complex
Article Snippet: Lysis buffer (25 mM HEPES-KOH pH 7.3, 100 mM KOAc, 5 mM MgOAc2 , 1 mM TCEP, 1 mM PMSF, Roche EDTA-free complete protease inhibitor) was added, and cells were detached with a cell scraper. .. For samples treated with RNase A, RNase A (Roche) was added to lysates (10 mg/mL final concentration) and incubated for 20 min at RT.

Article Title: Evidence that Lin28 stimulates translation by recruiting RNA helicase A to polysomes
Article Snippet: .. Cell pellet was resuspended in 400 µl of gentle lysis buffer [10 mM Tris–HCl at pH 7.5, 10 mM NaCl, 10 mM EDTA, 0.5% Triton X-100, 1 mM PMSF, 1× protease inhibitor cocktail (Calbiochem), 1 mM DTT and 10 µg/ml of RNase A (Roche)] and incubated on ice for 15 min. Insoluble materials were removed by centrifugation at 13 400 g in a microcentrifuge at 4°C for 15 min. NaCl was added to the cleared lysate to a final concentration of 200 mM, and 350 µl of the lysate incubated with 20 µl of protein-A sepharose beads pre-bound with 10 µl of anti-Lin28 antibody, pre-immune IgG ( C), or 10 µg of anti-Flag M2 antibody ( B) at 4°C overnight. .. The next day, beads were washed and bound fractions eluted by 3× SDS-sample buffer by heating at 95°C for 5 min. Proteins were resolved by SDS–PAGE, followed by western blot analysis.

Article Title: Genetic and Biochemical Assays Reveal a Key Role for Replication Restart Proteins in Group II Intron Retrohoming
Article Snippet: Assays were carried out in 20 µl of reaction mixture containing 50 nM DNA substrate, 3 µl of in vitro reconstituted Ll.LtrB RNPs (5–10 µg based on O.D.260 ; RNPs prepared as described in ref. ), 6 µl of S12 extract, 20 µM carrier DNA oligonucleotide ( 5′-GTGATGTCTGAAAAGAACGGGAAG ) as protection against DNase activity, 56.4 mM Tris-acetate buffer, pH 7.5, 100 mM potassium acetate, 35.9 mM ammonium acetate, 24 mM magnesium acetate, 1.5 mM ATP, 1 mM e ach of dATP, dCTP, dGTP, and dTTP (collectively denoted dNTPs), 500 µM of CTP, GTP, and UTP, 5 mM phosphoenolpyruvate, 50 µg/ml pyruvate kinase, 2 units/µl RNaseOUT (Invitrogen), and 1% (v/v) protease inhibitor cocktail [made by dissolving a mini EDTA-free tablet (Roche) in 1 ml of RNase-free water]. .. For RNase treatment, 0.4 units RNase H (Invitrogen) and 0.1 µg RNase A (Roche) were added, and the sample was incubated for 30 min at 37°C before the ethanol-precipitation step.

Article Title: Targeting and Anchoring Tudor in the Pole Plasm of the Drosophila Oocyte
Article Snippet: The supernatant was removed and the ovaries were suspended and homogenized in 125 µl of ice-cold IP buffer (145 mM NaCl, 10% glycerol, 1 mM MgCl2 , 1.5 mM NaH2 PO4 , 8 mM Na2 HPO4 [pH 7.4], 0.5% NP-40) containing protease inhibitors (complete EDTA-free protease inhibitor cocktail; Roche). .. For the release experiment the immuno-complexes were washed extensively and incubated for 20 minutes at room temperature with 50μg of RNase A (Roche).

Nick Translation:

Article Title: Copy number rather than epigenetic alterations are the major dictator of imprinted methylation in tumors
Article Snippet: Control slides were treated with RNAse A (Roche) treatment for 1 h at 37 °C and subsequently washed twice in 2× SSC. .. BAC probe RP11-937O11 (obtained from the BACPAC Resource Center) was labeled with either SpectrumRed or SpectrumGreen UTP by nick translation and competed with human COT-1 DNA (Roche) following the supplier’s protocol (Abbott Molecular Inc.).

Protein Concentration:

Article Title: Ternatin and improved synthetic variants kill cancer cells by targeting the elongation factor-1A ternary complex
Article Snippet: Crude lysates were clarified by centrifugation (15 min at 14,000 rpm), protein was quantified by Bradford assay, and the protein concentration was brought to 1.5 mg/mL with lysis buffer. .. For samples treated with RNase A, RNase A (Roche) was added to lysates (10 mg/mL final concentration) and incubated for 20 min at RT.

Reverse Transcription Polymerase Chain Reaction:

Article Title: Arf1p Provides an Unexpected Link between COPI Vesicles and mRNA in Saccharomyces cerevisiae D⃞
Article Snippet: Paragraph title: Coimmunoprecipitation Experiments and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) ... For RNA digestion experiments, the lysates were treated for 35 min at 4°C with 200 μg of RNase A (Roche Diagnostics, Indianapolis, IN) or 500 U of RNase-free DNase I and centrifuged before precipitation.

Affinity Purification:

Article Title: Arf1p Provides an Unexpected Link between COPI Vesicles and mRNA in Saccharomyces cerevisiae D⃞
Article Snippet: For Bfr1p coimmunoprecipitation, the lysate was incubated with 10 μl of affinity-purified anti-Arf1p antibody cross-linked to 25 μl of protein A magnetic beads (New England Biolabs, Beverly, MA) by using dimethyl pimelimidate (Pierce Chemical, Rockford, IL). .. For RNA digestion experiments, the lysates were treated for 35 min at 4°C with 200 μg of RNase A (Roche Diagnostics, Indianapolis, IN) or 500 U of RNase-free DNase I and centrifuged before precipitation.

Article Title: Modulation of dADAR-dependent RNA editing by the Drosophila fragile X mental retardation protein
Article Snippet: Paragraph title: Tandem affinity purification (TAP) assay for dADAR-TAP-expressing cells ... TAP purification was performed as previously described with the following modifications: After overnight incubation with IgG sepharose, beads were washed in TEV cleavage buffer five times, with the fourth wash being supplemented with RNase A (Roche, final concentration 0.05 μg/μl) for 1 hr.

Fluorescence:

Article Title: Dependence of Intracellular and Exosomal microRNAs on Viral E6/E7 Oncogene Expression in HPV-positive Tumor Cells
Article Snippet: Subsequently cells were pelleted, resuspended in phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2 HPO4 , 1.4 mM KH2 PO4 , pH 7.4) containing 1 mg/ml RNase A (Roche Diagnostics) and 25 μg/ml propidium iodide (Sigma-Aldrich) and incubated for 30 min at room temperature (RT). .. Cell cycle analyses were performed by fluorescence-activated cell sorting (FACS) using a FACSCalibur Flow Cytometer (BD Biosciences, Heidelberg, Germany) with CellQuest Pro software provided by the manufacturer.

Magnetic Beads:

Article Title: Arf1p Provides an Unexpected Link between COPI Vesicles and mRNA in Saccharomyces cerevisiae D⃞
Article Snippet: For Bfr1p coimmunoprecipitation, the lysate was incubated with 10 μl of affinity-purified anti-Arf1p antibody cross-linked to 25 μl of protein A magnetic beads (New England Biolabs, Beverly, MA) by using dimethyl pimelimidate (Pierce Chemical, Rockford, IL). .. For RNA digestion experiments, the lysates were treated for 35 min at 4°C with 200 μg of RNase A (Roche Diagnostics, Indianapolis, IN) or 500 U of RNase-free DNase I and centrifuged before precipitation.

Isolation:

Article Title: Dependence of Intracellular and Exosomal microRNAs on Viral E6/E7 Oncogene Expression in HPV-positive Tumor Cells
Article Snippet: .. Total exosomal RNA, including miRNA, was isolated using the protocol described for cells with slight modifications: Exosome samples were pre-treated with 100 ng/μl RNAse A (Roche) for 30 min at 37°C, immediately before extracting RNA. .. Prior to the addition of chloroform and phase separation, 12 μg glycogen from Mytilus edulis (Sigma) were added to the sample.

Article Title: Identification of Neuronal RNA Targets of TDP-43-containing Ribonucleoprotein Complexes *
Article Snippet: The pellet was suspended in nuclear isolation buffer (10 mm HEPES-NaOH, pH 7.4, 1 mm MgCl2 , 1.42 m sucrose, 1 mm DTT, 1× protease inhibitors), added to Beckman centrifuge tubes, and centrifuged in a SW 45 Ti rotor at 100,000 × g for 1 h. The nuclear pellet (P100) was suspended in NT2 lysis buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm MgCl2 , 0.05% Nonidet P-40, 20 mm DTT, 1× protease inhibitors). .. HeLa nuclear lysates were untreated or pretreated with RNase A (Roche Diagnostics) and rat brain nuclear lysates were untreated or pretreated with micrococcal nuclease before they were loaded into a Superose 6 or Superdex 200 column (GE Healthcare), respectively, in buffer containing 50 mm Tris-HCl, 150 mm NaCl at pH 7.5.

Flow Cytometry:

Article Title: Dependence of Intracellular and Exosomal microRNAs on Viral E6/E7 Oncogene Expression in HPV-positive Tumor Cells
Article Snippet: Subsequently cells were pelleted, resuspended in phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2 HPO4 , 1.4 mM KH2 PO4 , pH 7.4) containing 1 mg/ml RNase A (Roche Diagnostics) and 25 μg/ml propidium iodide (Sigma-Aldrich) and incubated for 30 min at room temperature (RT). .. Cell cycle analyses were performed by fluorescence-activated cell sorting (FACS) using a FACSCalibur Flow Cytometer (BD Biosciences, Heidelberg, Germany) with CellQuest Pro software provided by the manufacturer.

Labeling:

Article Title: Genetic and Biochemical Assays Reveal a Key Role for Replication Restart Proteins in Group II Intron Retrohoming
Article Snippet: Reactions were initiated by adding labeled DNA substrate, incubated at 37°C for times specified for individual experiments, and terminated by extraction with phenol-chloroform-isoamyl alcohol (phenol-CIA; 25∶24∶1 by volume). .. For RNase treatment, 0.4 units RNase H (Invitrogen) and 0.1 µg RNase A (Roche) were added, and the sample was incubated for 30 min at 37°C before the ethanol-precipitation step.

Article Title: Copy number rather than epigenetic alterations are the major dictator of imprinted methylation in tumors
Article Snippet: Control slides were treated with RNAse A (Roche) treatment for 1 h at 37 °C and subsequently washed twice in 2× SSC. .. BAC probe RP11-937O11 (obtained from the BACPAC Resource Center) was labeled with either SpectrumRed or SpectrumGreen UTP by nick translation and competed with human COT-1 DNA (Roche) following the supplier’s protocol (Abbott Molecular Inc.).

Purification:

Article Title: Modulation of dADAR-dependent RNA editing by the Drosophila fragile X mental retardation protein
Article Snippet: .. TAP purification was performed as previously described with the following modifications: After overnight incubation with IgG sepharose, beads were washed in TEV cleavage buffer five times, with the fourth wash being supplemented with RNase A (Roche, final concentration 0.05 μg/μl) for 1 hr. .. TEV cleavage was performed for 4 hrs and complexes were collected and run on SDS-PAGE followed by Western analysis.

FACS:

Article Title: Dependence of Intracellular and Exosomal microRNAs on Viral E6/E7 Oncogene Expression in HPV-positive Tumor Cells
Article Snippet: Subsequently cells were pelleted, resuspended in phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2 HPO4 , 1.4 mM KH2 PO4 , pH 7.4) containing 1 mg/ml RNase A (Roche Diagnostics) and 25 μg/ml propidium iodide (Sigma-Aldrich) and incubated for 30 min at room temperature (RT). .. Cell cycle analyses were performed by fluorescence-activated cell sorting (FACS) using a FACSCalibur Flow Cytometer (BD Biosciences, Heidelberg, Germany) with CellQuest Pro software provided by the manufacturer.

Construct:

Article Title: Modulation of dADAR-dependent RNA editing by the Drosophila fragile X mental retardation protein
Article Snippet: Tandem affinity purification (TAP) assay for dADAR-TAP-expressing cells For dADAR-TAP pulldown assays, Drosophila S2 cells expressing pCoBlast-dADAR-3A-TAP, pCoBlast-dADAR-3/4-TAP, or pCoBlast-TAP constructs were used for the TAP assay. .. TAP purification was performed as previously described with the following modifications: After overnight incubation with IgG sepharose, beads were washed in TEV cleavage buffer five times, with the fourth wash being supplemented with RNase A (Roche, final concentration 0.05 μg/μl) for 1 hr.

Cotransfection:

Article Title: Evidence that Lin28 stimulates translation by recruiting RNA helicase A to polysomes
Article Snippet: Co-immunoprecipitation To examine the interactions between Lin28 and RHA, 8 × 106 HEK293 (or PA-1) cells were transfected with 2 µg of Flag-Lin28 (Flag-28ΔC, Flag-28ΔN or empty vector), with or without co-transfection of 6 µg of Flag-N300 in a 6 cm plate scale (total DNA per plate was 8 µg). .. Cell pellet was resuspended in 400 µl of gentle lysis buffer [10 mM Tris–HCl at pH 7.5, 10 mM NaCl, 10 mM EDTA, 0.5% Triton X-100, 1 mM PMSF, 1× protease inhibitor cocktail (Calbiochem), 1 mM DTT and 10 µg/ml of RNase A (Roche)] and incubated on ice for 15 min. Insoluble materials were removed by centrifugation at 13 400 g in a microcentrifuge at 4°C for 15 min. NaCl was added to the cleared lysate to a final concentration of 200 mM, and 350 µl of the lysate incubated with 20 µl of protein-A sepharose beads pre-bound with 10 µl of anti-Lin28 antibody, pre-immune IgG ( C), or 10 µg of anti-Flag M2 antibody ( B) at 4°C overnight.

Lysis:

Article Title: Ternatin and improved synthetic variants kill cancer cells by targeting the elongation factor-1A ternary complex
Article Snippet: Crude lysates were clarified by centrifugation (15 min at 14,000 rpm), protein was quantified by Bradford assay, and the protein concentration was brought to 1.5 mg/mL with lysis buffer. .. For samples treated with RNase A, RNase A (Roche) was added to lysates (10 mg/mL final concentration) and incubated for 20 min at RT.

Article Title: Evidence that Lin28 stimulates translation by recruiting RNA helicase A to polysomes
Article Snippet: .. Cell pellet was resuspended in 400 µl of gentle lysis buffer [10 mM Tris–HCl at pH 7.5, 10 mM NaCl, 10 mM EDTA, 0.5% Triton X-100, 1 mM PMSF, 1× protease inhibitor cocktail (Calbiochem), 1 mM DTT and 10 µg/ml of RNase A (Roche)] and incubated on ice for 15 min. Insoluble materials were removed by centrifugation at 13 400 g in a microcentrifuge at 4°C for 15 min. NaCl was added to the cleared lysate to a final concentration of 200 mM, and 350 µl of the lysate incubated with 20 µl of protein-A sepharose beads pre-bound with 10 µl of anti-Lin28 antibody, pre-immune IgG ( C), or 10 µg of anti-Flag M2 antibody ( B) at 4°C overnight. .. The next day, beads were washed and bound fractions eluted by 3× SDS-sample buffer by heating at 95°C for 5 min. Proteins were resolved by SDS–PAGE, followed by western blot analysis.

Article Title: Identification of Neuronal RNA Targets of TDP-43-containing Ribonucleoprotein Complexes *
Article Snippet: The pellet was suspended in nuclear isolation buffer (10 mm HEPES-NaOH, pH 7.4, 1 mm MgCl2 , 1.42 m sucrose, 1 mm DTT, 1× protease inhibitors), added to Beckman centrifuge tubes, and centrifuged in a SW 45 Ti rotor at 100,000 × g for 1 h. The nuclear pellet (P100) was suspended in NT2 lysis buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm MgCl2 , 0.05% Nonidet P-40, 20 mm DTT, 1× protease inhibitors). .. HeLa nuclear lysates were untreated or pretreated with RNase A (Roche Diagnostics) and rat brain nuclear lysates were untreated or pretreated with micrococcal nuclease before they were loaded into a Superose 6 or Superdex 200 column (GE Healthcare), respectively, in buffer containing 50 mm Tris-HCl, 150 mm NaCl at pH 7.5.

Concentration Assay:

Article Title: HIV Tat controls RNA Polymerase II and the epigenetic landscape to transcriptionally reprogram target immune cells
Article Snippet: .. The pellet was similarly resuspended in 2× pellet volume of buffer B containing 600 mM NaCl, vortexed for 10 s, treated with RNAse A (Roche) or BSA (mock treatment) at a concentration of 10 μg/ml for 60 min at 4°C and then spun at 6000 g for 5 min. .. Western blotting and antibodies Protein samples were resolved in sodium dodecyl sufate (SDS)-polyacrylamide gel electrophoresis, transferred to 0.45 μm nitrocellulose (Bio-Rad, Hercules, CA) membranes, blocked in Tris-buffered saline (TBS) containing 5% non-fat dry milk for 1 hr, and incubated with primary antibodies overnight at 4°C.

Article Title: Ternatin and improved synthetic variants kill cancer cells by targeting the elongation factor-1A ternary complex
Article Snippet: .. For samples treated with RNase A, RNase A (Roche) was added to lysates (10 mg/mL final concentration) and incubated for 20 min at RT. ..

Article Title: Evidence that Lin28 stimulates translation by recruiting RNA helicase A to polysomes
Article Snippet: .. Cell pellet was resuspended in 400 µl of gentle lysis buffer [10 mM Tris–HCl at pH 7.5, 10 mM NaCl, 10 mM EDTA, 0.5% Triton X-100, 1 mM PMSF, 1× protease inhibitor cocktail (Calbiochem), 1 mM DTT and 10 µg/ml of RNase A (Roche)] and incubated on ice for 15 min. Insoluble materials were removed by centrifugation at 13 400 g in a microcentrifuge at 4°C for 15 min. NaCl was added to the cleared lysate to a final concentration of 200 mM, and 350 µl of the lysate incubated with 20 µl of protein-A sepharose beads pre-bound with 10 µl of anti-Lin28 antibody, pre-immune IgG ( C), or 10 µg of anti-Flag M2 antibody ( B) at 4°C overnight. .. The next day, beads were washed and bound fractions eluted by 3× SDS-sample buffer by heating at 95°C for 5 min. Proteins were resolved by SDS–PAGE, followed by western blot analysis.

Article Title: Ribonucleoprotein Particles Containing Non-Coding Y RNAs, Ro60, La and Nucleolin Are Not Required for Y RNA Function in DNA Replication
Article Snippet: .. Extract was diluted in replication buffer (20 mM K-HEPES pH 7.8, 100 mM K-acetate, 1 mM DTT, 1 mM EGTA) and pre-depleted with protein G-agarose or protein A-agarose (both from Roche). (Where applicable, pre-depleted extract was treated with a final concentration of 0.3 mg/ml RNase A (Roche).) .. Pre-depleted extracts were incubated with the following primary antibodies: anti-60 (Euro-Diagnostica), anti-La (Santa Cruz sc-80655), and anti-nucleolin (a gift from Professor Hans Stahl, University of Homburg/Saar.

Article Title: Modulation of dADAR-dependent RNA editing by the Drosophila fragile X mental retardation protein
Article Snippet: .. TAP purification was performed as previously described with the following modifications: After overnight incubation with IgG sepharose, beads were washed in TEV cleavage buffer five times, with the fourth wash being supplemented with RNase A (Roche, final concentration 0.05 μg/μl) for 1 hr. .. TEV cleavage was performed for 4 hrs and complexes were collected and run on SDS-PAGE followed by Western analysis.

SDS Page:

Article Title: Evidence that Lin28 stimulates translation by recruiting RNA helicase A to polysomes
Article Snippet: Cell pellet was resuspended in 400 µl of gentle lysis buffer [10 mM Tris–HCl at pH 7.5, 10 mM NaCl, 10 mM EDTA, 0.5% Triton X-100, 1 mM PMSF, 1× protease inhibitor cocktail (Calbiochem), 1 mM DTT and 10 µg/ml of RNase A (Roche)] and incubated on ice for 15 min. Insoluble materials were removed by centrifugation at 13 400 g in a microcentrifuge at 4°C for 15 min. NaCl was added to the cleared lysate to a final concentration of 200 mM, and 350 µl of the lysate incubated with 20 µl of protein-A sepharose beads pre-bound with 10 µl of anti-Lin28 antibody, pre-immune IgG ( C), or 10 µg of anti-Flag M2 antibody ( B) at 4°C overnight. .. The next day, beads were washed and bound fractions eluted by 3× SDS-sample buffer by heating at 95°C for 5 min. Proteins were resolved by SDS–PAGE, followed by western blot analysis.

Article Title: Modulation of dADAR-dependent RNA editing by the Drosophila fragile X mental retardation protein
Article Snippet: TAP purification was performed as previously described with the following modifications: After overnight incubation with IgG sepharose, beads were washed in TEV cleavage buffer five times, with the fourth wash being supplemented with RNase A (Roche, final concentration 0.05 μg/μl) for 1 hr. .. TEV cleavage was performed for 4 hrs and complexes were collected and run on SDS-PAGE followed by Western analysis.

Plasmid Preparation:

Article Title: Evidence that Lin28 stimulates translation by recruiting RNA helicase A to polysomes
Article Snippet: Co-immunoprecipitation To examine the interactions between Lin28 and RHA, 8 × 106 HEK293 (or PA-1) cells were transfected with 2 µg of Flag-Lin28 (Flag-28ΔC, Flag-28ΔN or empty vector), with or without co-transfection of 6 µg of Flag-N300 in a 6 cm plate scale (total DNA per plate was 8 µg). .. Cell pellet was resuspended in 400 µl of gentle lysis buffer [10 mM Tris–HCl at pH 7.5, 10 mM NaCl, 10 mM EDTA, 0.5% Triton X-100, 1 mM PMSF, 1× protease inhibitor cocktail (Calbiochem), 1 mM DTT and 10 µg/ml of RNase A (Roche)] and incubated on ice for 15 min. Insoluble materials were removed by centrifugation at 13 400 g in a microcentrifuge at 4°C for 15 min. NaCl was added to the cleared lysate to a final concentration of 200 mM, and 350 µl of the lysate incubated with 20 µl of protein-A sepharose beads pre-bound with 10 µl of anti-Lin28 antibody, pre-immune IgG ( C), or 10 µg of anti-Flag M2 antibody ( B) at 4°C overnight.

Software:

Article Title: Dependence of Intracellular and Exosomal microRNAs on Viral E6/E7 Oncogene Expression in HPV-positive Tumor Cells
Article Snippet: Subsequently cells were pelleted, resuspended in phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2 HPO4 , 1.4 mM KH2 PO4 , pH 7.4) containing 1 mg/ml RNase A (Roche Diagnostics) and 25 μg/ml propidium iodide (Sigma-Aldrich) and incubated for 30 min at room temperature (RT). .. Cell cycle analyses were performed by fluorescence-activated cell sorting (FACS) using a FACSCalibur Flow Cytometer (BD Biosciences, Heidelberg, Germany) with CellQuest Pro software provided by the manufacturer.

RNA Extraction:

Article Title: Dependence of Intracellular and Exosomal microRNAs on Viral E6/E7 Oncogene Expression in HPV-positive Tumor Cells
Article Snippet: Paragraph title: RNA extraction, quantification and quality determination ... Total exosomal RNA, including miRNA, was isolated using the protocol described for cells with slight modifications: Exosome samples were pre-treated with 100 ng/μl RNAse A (Roche) for 30 min at 37°C, immediately before extracting RNA.

Agarose Gel Electrophoresis:

Article Title: Genetic and Biochemical Assays Reveal a Key Role for Replication Restart Proteins in Group II Intron Retrohoming
Article Snippet: For RNase treatment, 0.4 units RNase H (Invitrogen) and 0.1 µg RNase A (Roche) were added, and the sample was incubated for 30 min at 37°C before the ethanol-precipitation step. .. For primer-extension analysis, DNA products were separated in a 1% low-melting point agarose gel (Fermentas).

In Vitro:

Article Title: Determinants of R-loop formation at convergent bidirectionally transcribed trinucleotide repeats
Article Snippet: .. RNase treatment and electrophoresis To analyze hybrid formation, samples from in vitro transcription reactions were divided into three (5 μl each) and treated with either TE (transcription control), 1 μg RNase A (Roche) or 10U of RNase T1 (Roche), 1 μg RNase A + 1 U RNase H (Roche) or 10U RNase T1 + 1 U RNase H as stated in a final volume of 10 μl for 20 min at room temperature. .. All in vitro transcription reaction products were analyzed on 0.8% agarose gels run in 1× Tris–Borate–EDTA buffer at 80 V for 5 h. Gels were subsequently stained with ethidium bromide (0.5 μg/ml) to allow visualization of the nucleic acid products under UV light.

Article Title: Genetic and Biochemical Assays Reveal a Key Role for Replication Restart Proteins in Group II Intron Retrohoming
Article Snippet: Assays were carried out in 20 µl of reaction mixture containing 50 nM DNA substrate, 3 µl of in vitro reconstituted Ll.LtrB RNPs (5–10 µg based on O.D.260 ; RNPs prepared as described in ref. ), 6 µl of S12 extract, 20 µM carrier DNA oligonucleotide ( 5′-GTGATGTCTGAAAAGAACGGGAAG ) as protection against DNase activity, 56.4 mM Tris-acetate buffer, pH 7.5, 100 mM potassium acetate, 35.9 mM ammonium acetate, 24 mM magnesium acetate, 1.5 mM ATP, 1 mM e ach of dATP, dCTP, dGTP, and dTTP (collectively denoted dNTPs), 500 µM of CTP, GTP, and UTP, 5 mM phosphoenolpyruvate, 50 µg/ml pyruvate kinase, 2 units/µl RNaseOUT (Invitrogen), and 1% (v/v) protease inhibitor cocktail [made by dissolving a mini EDTA-free tablet (Roche) in 1 ml of RNase-free water]. .. For RNase treatment, 0.4 units RNase H (Invitrogen) and 0.1 µg RNase A (Roche) were added, and the sample was incubated for 30 min at 37°C before the ethanol-precipitation step.

Size-exclusion Chromatography:

Article Title: Identification of Neuronal RNA Targets of TDP-43-containing Ribonucleoprotein Complexes *
Article Snippet: Paragraph title: TDP-43 Co-immunoprecipitation and Size Exclusion Chromatography ... HeLa nuclear lysates were untreated or pretreated with RNase A (Roche Diagnostics) and rat brain nuclear lysates were untreated or pretreated with micrococcal nuclease before they were loaded into a Superose 6 or Superdex 200 column (GE Healthcare), respectively, in buffer containing 50 mm Tris-HCl, 150 mm NaCl at pH 7.5.

Ethanol Precipitation:

Article Title: Genetic and Biochemical Assays Reveal a Key Role for Replication Restart Proteins in Group II Intron Retrohoming
Article Snippet: .. For RNase treatment, 0.4 units RNase H (Invitrogen) and 0.1 µg RNase A (Roche) were added, and the sample was incubated for 30 min at 37°C before the ethanol-precipitation step. ..

Incubation:

Article Title: Arf1p Provides an Unexpected Link between COPI Vesicles and mRNA in Saccharomyces cerevisiae D⃞
Article Snippet: For Bfr1p coimmunoprecipitation, the lysate was incubated with 10 μl of affinity-purified anti-Arf1p antibody cross-linked to 25 μl of protein A magnetic beads (New England Biolabs, Beverly, MA) by using dimethyl pimelimidate (Pierce Chemical, Rockford, IL). .. For RNA digestion experiments, the lysates were treated for 35 min at 4°C with 200 μg of RNase A (Roche Diagnostics, Indianapolis, IN) or 500 U of RNase-free DNase I and centrifuged before precipitation.

Article Title: HIV Tat controls RNA Polymerase II and the epigenetic landscape to transcriptionally reprogram target immune cells
Article Snippet: The cell pellet (PCV = 0.1 ml) was resuspended in 1 ml buffer A (10 mM KCl, 10 mM HEPES pH 7.9, 0.1 mM EDTA, 1 mM DTT, 0.4% NP-40, plus PI) and incubated on ice for 5 min. .. The pellet was similarly resuspended in 2× pellet volume of buffer B containing 600 mM NaCl, vortexed for 10 s, treated with RNAse A (Roche) or BSA (mock treatment) at a concentration of 10 μg/ml for 60 min at 4°C and then spun at 6000 g for 5 min.

Article Title: Ternatin and improved synthetic variants kill cancer cells by targeting the elongation factor-1A ternary complex
Article Snippet: .. For samples treated with RNase A, RNase A (Roche) was added to lysates (10 mg/mL final concentration) and incubated for 20 min at RT. ..

Article Title: Dependence of Intracellular and Exosomal microRNAs on Viral E6/E7 Oncogene Expression in HPV-positive Tumor Cells
Article Snippet: .. Subsequently cells were pelleted, resuspended in phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2 HPO4 , 1.4 mM KH2 PO4 , pH 7.4) containing 1 mg/ml RNase A (Roche Diagnostics) and 25 μg/ml propidium iodide (Sigma-Aldrich) and incubated for 30 min at room temperature (RT). .. Cell cycle analyses were performed by fluorescence-activated cell sorting (FACS) using a FACSCalibur Flow Cytometer (BD Biosciences, Heidelberg, Germany) with CellQuest Pro software provided by the manufacturer.

Article Title: Evidence that Lin28 stimulates translation by recruiting RNA helicase A to polysomes
Article Snippet: .. Cell pellet was resuspended in 400 µl of gentle lysis buffer [10 mM Tris–HCl at pH 7.5, 10 mM NaCl, 10 mM EDTA, 0.5% Triton X-100, 1 mM PMSF, 1× protease inhibitor cocktail (Calbiochem), 1 mM DTT and 10 µg/ml of RNase A (Roche)] and incubated on ice for 15 min. Insoluble materials were removed by centrifugation at 13 400 g in a microcentrifuge at 4°C for 15 min. NaCl was added to the cleared lysate to a final concentration of 200 mM, and 350 µl of the lysate incubated with 20 µl of protein-A sepharose beads pre-bound with 10 µl of anti-Lin28 antibody, pre-immune IgG ( C), or 10 µg of anti-Flag M2 antibody ( B) at 4°C overnight. .. The next day, beads were washed and bound fractions eluted by 3× SDS-sample buffer by heating at 95°C for 5 min. Proteins were resolved by SDS–PAGE, followed by western blot analysis.

Article Title: Genetic and Biochemical Assays Reveal a Key Role for Replication Restart Proteins in Group II Intron Retrohoming
Article Snippet: .. For RNase treatment, 0.4 units RNase H (Invitrogen) and 0.1 µg RNase A (Roche) were added, and the sample was incubated for 30 min at 37°C before the ethanol-precipitation step. ..

Article Title: HIV Tat controls RNA Polymerase II and the epigenetic landscape to transcriptionally reprogram target immune cells
Article Snippet: Reverse crosslinking was done by incubation with 25 μl (20 mg/ml) of proteinase K (Roche) at 65°C overnight. .. DNA samples were then treated with 5 μl of RNase A (Roche) for 30 min at 37°C, and then mixed with 7 ml of phenol–chloroform vigorously and centrifuged for 15 min at 2200 g at room temperature.

Article Title: Ribonucleoprotein Particles Containing Non-Coding Y RNAs, Ro60, La and Nucleolin Are Not Required for Y RNA Function in DNA Replication
Article Snippet: Extract was diluted in replication buffer (20 mM K-HEPES pH 7.8, 100 mM K-acetate, 1 mM DTT, 1 mM EGTA) and pre-depleted with protein G-agarose or protein A-agarose (both from Roche). (Where applicable, pre-depleted extract was treated with a final concentration of 0.3 mg/ml RNase A (Roche).) .. Pre-depleted extracts were incubated with the following primary antibodies: anti-60 (Euro-Diagnostica), anti-La (Santa Cruz sc-80655), and anti-nucleolin (a gift from Professor Hans Stahl, University of Homburg/Saar.

Article Title: Targeting and Anchoring Tudor in the Pole Plasm of the Drosophila Oocyte
Article Snippet: .. For the release experiment the immuno-complexes were washed extensively and incubated for 20 minutes at room temperature with 50μg of RNase A (Roche). .. Immunocytochemistry Ovaries were dissected in PBS, fixed in 4% paraformaldehyde:heptane (1∶1) in PBS for 12 minutes, washed four times for 20 min in 0.1% Tween 20 in PBS (PBT), treated one hour in 1% Triton X-100 in PBS, and blocked for 1 h in 2% BSA in PBT.

Article Title: Identification of Neuronal RNA Targets of TDP-43-containing Ribonucleoprotein Complexes *
Article Snippet: Lysates were incubated overnight at 4 °C and eluted with 50 mm glycine, pH 2.5, and eluents were neutralized with 1 m Tris-HCl, pH 8.0. .. HeLa nuclear lysates were untreated or pretreated with RNase A (Roche Diagnostics) and rat brain nuclear lysates were untreated or pretreated with micrococcal nuclease before they were loaded into a Superose 6 or Superdex 200 column (GE Healthcare), respectively, in buffer containing 50 mm Tris-HCl, 150 mm NaCl at pH 7.5.

Article Title: Modulation of dADAR-dependent RNA editing by the Drosophila fragile X mental retardation protein
Article Snippet: .. TAP purification was performed as previously described with the following modifications: After overnight incubation with IgG sepharose, beads were washed in TEV cleavage buffer five times, with the fourth wash being supplemented with RNase A (Roche, final concentration 0.05 μg/μl) for 1 hr. .. TEV cleavage was performed for 4 hrs and complexes were collected and run on SDS-PAGE followed by Western analysis.

Immunoprecipitation:

Article Title: Ribonucleoprotein Particles Containing Non-Coding Y RNAs, Ro60, La and Nucleolin Are Not Required for Y RNA Function in DNA Replication
Article Snippet: Paragraph title: Immunoprecipitation ... Extract was diluted in replication buffer (20 mM K-HEPES pH 7.8, 100 mM K-acetate, 1 mM DTT, 1 mM EGTA) and pre-depleted with protein G-agarose or protein A-agarose (both from Roche). (Where applicable, pre-depleted extract was treated with a final concentration of 0.3 mg/ml RNase A (Roche).)

Article Title: Targeting and Anchoring Tudor in the Pole Plasm of the Drosophila Oocyte
Article Snippet: Paragraph title: Immuno-precipitation ... For the release experiment the immuno-complexes were washed extensively and incubated for 20 minutes at room temperature with 50μg of RNase A (Roche).

BAC Assay:

Article Title: HIV Tat controls RNA Polymerase II and the epigenetic landscape to transcriptionally reprogram target immune cells
Article Snippet: DNA samples were then treated with 5 μl of RNase A (Roche) for 30 min at 37°C, and then mixed with 7 ml of phenol–chloroform vigorously and centrifuged for 15 min at 2200 g at room temperature. .. Random ligation matrix was prepared by digestion with restriction enzyme and ligation with BAC clones containing the respective genes examined from the Children’s Hospital Oakland Research Institute (CHORI, Oakland, CA).

Article Title: Copy number rather than epigenetic alterations are the major dictator of imprinted methylation in tumors
Article Snippet: Control slides were treated with RNAse A (Roche) treatment for 1 h at 37 °C and subsequently washed twice in 2× SSC. .. BAC probe RP11-937O11 (obtained from the BACPAC Resource Center) was labeled with either SpectrumRed or SpectrumGreen UTP by nick translation and competed with human COT-1 DNA (Roche) following the supplier’s protocol (Abbott Molecular Inc.).

Staining:

Article Title: Determinants of R-loop formation at convergent bidirectionally transcribed trinucleotide repeats
Article Snippet: RNase treatment and electrophoresis To analyze hybrid formation, samples from in vitro transcription reactions were divided into three (5 μl each) and treated with either TE (transcription control), 1 μg RNase A (Roche) or 10U of RNase T1 (Roche), 1 μg RNase A + 1 U RNase H (Roche) or 10U RNase T1 + 1 U RNase H as stated in a final volume of 10 μl for 20 min at room temperature. .. All in vitro transcription reaction products were analyzed on 0.8% agarose gels run in 1× Tris–Borate–EDTA buffer at 80 V for 5 h. Gels were subsequently stained with ethidium bromide (0.5 μg/ml) to allow visualization of the nucleic acid products under UV light.

Fluorescence In Situ Hybridization:

Article Title: Copy number rather than epigenetic alterations are the major dictator of imprinted methylation in tumors
Article Snippet: Paragraph title: RNA FISH ... Control slides were treated with RNAse A (Roche) treatment for 1 h at 37 °C and subsequently washed twice in 2× SSC.

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 82
    Roche mouse rnase4
    Effect of <t>RNASE4</t> on SOD1 G93A mice. Starting from 11 weeks of age, mice were treated with weekly i.p. injection of WT RNASE4 protein at 10 μg per mouse. Three independent experiments were performed with a total of 34 and 31 mice in the RNASE4 treatment and PBS control group, respectively. a Effect on rotarod performance at 20 rpm without revolving. Two successive measurements were recorded. An upper limit of 1,000 second was used. b Effect on body weight. Data shown are means ± SEM of all survived animals at each data point. Statistical analysis was performed by two-way ANOVA.*, p
    Mouse Rnase4, supplied by Roche, used in various techniques. Bioz Stars score: 82/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse rnase4/product/Roche
    Average 82 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse rnase4 - by Bioz Stars, 2020-01
    82/100 stars
      Buy from Supplier

    94
    Roche recombinant human rnase h2
    Related to Figure 2j. RNASEH2A P40D/Y210A is a separation-of-function mutant that cannot excise single DNA-embedded ribonucleotides, but cleaves RNA:DNA heteroduplexes (similar to the yeast rnh201-P45D-Y219A mutant 16 ). a, Schematic depicting enzymatic activity against two different <t>RNase</t> H2 substrates (DRD:DNA, dsDNA with embedded ribonucleotide, or RNA:DNA hybrids) in cell lines used in b-d and Fig 2j . WT and RNASEH2A-KO cells were transduced with either an empty vector (EV) or the indicated RNASEH2A constructs. b, Complementation of HeLa RNASEH2A-KO cells with FLAG-tagged RNASEH2A variants restores RNase H2 complex protein levels. WCEs from HeLa WT and RNASEH2A-KO cells stably expressing indicated lentiviral constructs were processed for immunoblotting with the indicated antibodies. Vinculin, loading control. Asterisk indicates a non-specific band. Representative of n = 3 biologically independent experiments. c , d, Complementation of HeLa RNASEH2A-KO cells with WT RNASEH2A, but not with the D34A/D169A (catalytic-dead) or P40D/Y210A (separation-of-function) mutants, rescues increased levels of genome-embedded ribonucleotides. c , Total nucleic acids from the cell lines shown in a , b were treated with recombinant RNase H2 and separated by alkaline agarose gel electrophoresis (representative of n = 4 experiments). d, Densitometric quantification of alkaline gel shown in c . e , Purified human RNase H2 complexes consisting of RNASEH2B, RNASEH2C and either RNASEH2A WT, P40D/Y210A or D34A/D169A subunits separated by SDS-PAGE and stained with Coomassie Blue ( n = 1). f-k , RNase H2 activity assays with fluorescein-labeled RNA:DNA substrate ( f ) or double-stranded DNA with a single incorporated ribonucleotide (DRD:DNA) ( g ) and increasing amounts of recombinant WT, P40D/Y210A or D34A/D169A RNase H2. Products were separated by polyacrylamide gel electrophoresis and detected by fluorescence imaging. Representative of n = 3 biologically independent experiments. h,k, Quantification of f , g . Product signal plotted relative to substrate signal per lane. Mean ±SD ( n = 3 biologically independent experiments).
    Recombinant Human Rnase H2, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant human rnase h2/product/Roche
    Average 94 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    recombinant human rnase h2 - by Bioz Stars, 2020-01
    94/100 stars
      Buy from Supplier

    78
    Roche rnase l expression plasmid
    Expression of <t>RNase</t> L blocks L1 RNP formation. HeLa-M cells were co-transfected with pES2TE1 and either an empty vector (pcDNA 3.0) or a plasmid that encodes an amino-terminal Myc-tagged RNase L expression plasmid. Immunofluorescent confocal microscopy was used to examine L1 ORF2p accumulation in cytoplasmic foci by exploiting the FLAG-HA epitope-tag in pES2TE1 48 h after transfection. The top labels indicate the antibodies used to detect the indicated proteins: anti-HA-ORF2p, red; anti-EBNA-1, green; anti-Myc RNase L, magenta. The labels on the left side of the figure indicate the empty vector or RNase L constructs that were co-transfected into cells. The rightmost column indicates the merged overlay staining. L1 ORF2p formed discrete cytoplasmic punctate localization in co-transfection experiments performed with the empty vector and RNase L catalytically inactive mutant (R667A), but not with WT RNase L. For each condition, either two or three slides were examined per experiment. About 200 cells were examined per slide and representative images were captured. The experiment was conducted three times (biological replicates) with similar results.
    Rnase L Expression Plasmid, supplied by Roche, used in various techniques. Bioz Stars score: 78/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnase l expression plasmid/product/Roche
    Average 78 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rnase l expression plasmid - by Bioz Stars, 2020-01
    78/100 stars
      Buy from Supplier

    Image Search Results


    Effect of RNASE4 on SOD1 G93A mice. Starting from 11 weeks of age, mice were treated with weekly i.p. injection of WT RNASE4 protein at 10 μg per mouse. Three independent experiments were performed with a total of 34 and 31 mice in the RNASE4 treatment and PBS control group, respectively. a Effect on rotarod performance at 20 rpm without revolving. Two successive measurements were recorded. An upper limit of 1,000 second was used. b Effect on body weight. Data shown are means ± SEM of all survived animals at each data point. Statistical analysis was performed by two-way ANOVA.*, p

    Journal: Angiogenesis

    Article Title: Ribonuclease 4 protects neuron degeneration by promoting angiogenesis, neurogenesis, and neuronal survival under stress

    doi: 10.1007/s10456-012-9322-9

    Figure Lengend Snippet: Effect of RNASE4 on SOD1 G93A mice. Starting from 11 weeks of age, mice were treated with weekly i.p. injection of WT RNASE4 protein at 10 μg per mouse. Three independent experiments were performed with a total of 34 and 31 mice in the RNASE4 treatment and PBS control group, respectively. a Effect on rotarod performance at 20 rpm without revolving. Two successive measurements were recorded. An upper limit of 1,000 second was used. b Effect on body weight. Data shown are means ± SEM of all survived animals at each data point. Statistical analysis was performed by two-way ANOVA.*, p

    Article Snippet: Mouse Rnase4, forward, 5′- GGGTAATACGACTCACTATAGGGCGAtccgggtccaggcactttcta-3′; reverse, 5′-gtg ctggttcttgccctgtatcta-3′. cRNA probes were produced by in vitro transcription with T7 RNA polymerase from 1 μg of the above purified PCR products as template and were labeled with digoxigenin (Roche) per manufacturer’s protocol.

    Techniques: Mouse Assay, Injection

    Decreased RNASE4 mRNA level in the spinal cord motor neurons of ALS patients and in SOD1 G93A mice. a In situ hybridization of human RNASE4 mRNA in the spinal cord of ALS patients and non-ALS control subject. Left panel, representative ISH images from one of the six patients. Bar, 10 μm. Right panel, ImageJ analysis of photon counts per motor neuron. Data shown are means ± SEM from six patients. Statistical analysis was performed by two-way ANOVA. b In situ hybridization of mouse Rnase4 mRNA in the spinal cord of WT and SOD1 G93A mice. Left panel, representative ISH images from one of the six mice. Bar, 10 μm. Right panel, ImageJ analysis of photon counts per motor neuron. Data shown are means ± SEM from six patients. Statistical analysis was performed by two-way ANOVA.

    Journal: Angiogenesis

    Article Title: Ribonuclease 4 protects neuron degeneration by promoting angiogenesis, neurogenesis, and neuronal survival under stress

    doi: 10.1007/s10456-012-9322-9

    Figure Lengend Snippet: Decreased RNASE4 mRNA level in the spinal cord motor neurons of ALS patients and in SOD1 G93A mice. a In situ hybridization of human RNASE4 mRNA in the spinal cord of ALS patients and non-ALS control subject. Left panel, representative ISH images from one of the six patients. Bar, 10 μm. Right panel, ImageJ analysis of photon counts per motor neuron. Data shown are means ± SEM from six patients. Statistical analysis was performed by two-way ANOVA. b In situ hybridization of mouse Rnase4 mRNA in the spinal cord of WT and SOD1 G93A mice. Left panel, representative ISH images from one of the six mice. Bar, 10 μm. Right panel, ImageJ analysis of photon counts per motor neuron. Data shown are means ± SEM from six patients. Statistical analysis was performed by two-way ANOVA.

    Article Snippet: Mouse Rnase4, forward, 5′- GGGTAATACGACTCACTATAGGGCGAtccgggtccaggcactttcta-3′; reverse, 5′-gtg ctggttcttgccctgtatcta-3′. cRNA probes were produced by in vitro transcription with T7 RNA polymerase from 1 μg of the above purified PCR products as template and were labeled with digoxigenin (Roche) per manufacturer’s protocol.

    Techniques: Mouse Assay, In Situ Hybridization

    RNASE4 protects stress-induced neuron degeneration. a Effect on hypothermia-induced neurofilament fragmentation of mouse cortical neurons. Mouse cortical neurons were cultured in the presence of B27 for 12 days. Cells were washed with neurobasal medium, incubated with 0.2 μg/ml RNASE4 or ANG at 37 °C for 1 hour, and then subjected to hypothermia treatment at 25 °C for 40 min. Cells were returned to incubator and continually cultured for 3 h and stained for neurofilaments. The images are representative of at least four areas from three independent experiments. Bar, 0.4 mm. b ImageJ analysis of the length of neurofilament. Data shown are means ± SEM of three independent experiments in triplicates. Statistical analysis was performed by two-way ANOVA. **, p

    Journal: Angiogenesis

    Article Title: Ribonuclease 4 protects neuron degeneration by promoting angiogenesis, neurogenesis, and neuronal survival under stress

    doi: 10.1007/s10456-012-9322-9

    Figure Lengend Snippet: RNASE4 protects stress-induced neuron degeneration. a Effect on hypothermia-induced neurofilament fragmentation of mouse cortical neurons. Mouse cortical neurons were cultured in the presence of B27 for 12 days. Cells were washed with neurobasal medium, incubated with 0.2 μg/ml RNASE4 or ANG at 37 °C for 1 hour, and then subjected to hypothermia treatment at 25 °C for 40 min. Cells were returned to incubator and continually cultured for 3 h and stained for neurofilaments. The images are representative of at least four areas from three independent experiments. Bar, 0.4 mm. b ImageJ analysis of the length of neurofilament. Data shown are means ± SEM of three independent experiments in triplicates. Statistical analysis was performed by two-way ANOVA. **, p

    Article Snippet: Mouse Rnase4, forward, 5′- GGGTAATACGACTCACTATAGGGCGAtccgggtccaggcactttcta-3′; reverse, 5′-gtg ctggttcttgccctgtatcta-3′. cRNA probes were produced by in vitro transcription with T7 RNA polymerase from 1 μg of the above purified PCR products as template and were labeled with digoxigenin (Roche) per manufacturer’s protocol.

    Techniques: Cell Culture, Incubation, Staining

    Subcellular localization of RNASE4 in HUVE and P19 cells. a HUVE cells were incubated in the absence (top panel) or presence (bottom panel) of 0.5 μg/ml exogenous RNASE4 protein at 37 °C for 1 h. b P19 cells were incubated in the absence (top panel) or presence (bottom panel) of 1 μg/ml exogenous RNASE4 protein at 37 °C for 1 h. Immunofluorescence was carried out with affinity-purified RNASE4 polyclonal rabbit IgG and Alexa 488-labeld goat anti-rabbit IgG. Nuclei were stained with DAPI. Scale bar, 10 μm.

    Journal: Angiogenesis

    Article Title: Ribonuclease 4 protects neuron degeneration by promoting angiogenesis, neurogenesis, and neuronal survival under stress

    doi: 10.1007/s10456-012-9322-9

    Figure Lengend Snippet: Subcellular localization of RNASE4 in HUVE and P19 cells. a HUVE cells were incubated in the absence (top panel) or presence (bottom panel) of 0.5 μg/ml exogenous RNASE4 protein at 37 °C for 1 h. b P19 cells were incubated in the absence (top panel) or presence (bottom panel) of 1 μg/ml exogenous RNASE4 protein at 37 °C for 1 h. Immunofluorescence was carried out with affinity-purified RNASE4 polyclonal rabbit IgG and Alexa 488-labeld goat anti-rabbit IgG. Nuclei were stained with DAPI. Scale bar, 10 μm.

    Article Snippet: Mouse Rnase4, forward, 5′- GGGTAATACGACTCACTATAGGGCGAtccgggtccaggcactttcta-3′; reverse, 5′-gtg ctggttcttgccctgtatcta-3′. cRNA probes were produced by in vitro transcription with T7 RNA polymerase from 1 μg of the above purified PCR products as template and were labeled with digoxigenin (Roche) per manufacturer’s protocol.

    Techniques: Incubation, Immunofluorescence, Affinity Purification, Staining

    Effect of RNASE4 on neuronal differentiation. a RNASE4 stimulates neurosphere formation of P19 cells. P19 cells were cultured on PA6 supporting cell layers in the presence of 0.2 μg/ml of BSA, RNASE4, or ANG for 24 h. The images are representative of at least four areas from three independent experiments. Bar, 0.5 mm. b Numbers of neurosphere counted from the entire 35-mm dish. Data shown are means ± SEM of three independent experiments in triplicates. Statistical analysis was performed by two-way ANOVA. *, p

    Journal: Angiogenesis

    Article Title: Ribonuclease 4 protects neuron degeneration by promoting angiogenesis, neurogenesis, and neuronal survival under stress

    doi: 10.1007/s10456-012-9322-9

    Figure Lengend Snippet: Effect of RNASE4 on neuronal differentiation. a RNASE4 stimulates neurosphere formation of P19 cells. P19 cells were cultured on PA6 supporting cell layers in the presence of 0.2 μg/ml of BSA, RNASE4, or ANG for 24 h. The images are representative of at least four areas from three independent experiments. Bar, 0.5 mm. b Numbers of neurosphere counted from the entire 35-mm dish. Data shown are means ± SEM of three independent experiments in triplicates. Statistical analysis was performed by two-way ANOVA. *, p

    Article Snippet: Mouse Rnase4, forward, 5′- GGGTAATACGACTCACTATAGGGCGAtccgggtccaggcactttcta-3′; reverse, 5′-gtg ctggttcttgccctgtatcta-3′. cRNA probes were produced by in vitro transcription with T7 RNA polymerase from 1 μg of the above purified PCR products as template and were labeled with digoxigenin (Roche) per manufacturer’s protocol.

    Techniques: Cell Culture

    Ribonucleolytic activity of recombinant wide type RNASE4 and the K40A variant. a SDS-PAGE and Coomassie blue staining of WT and K40A RNASE4 from one of the four preparations. Recombinant ANG protein was included as a control. b Ribonuclolysis of yeast tRNA by WT RNASE4 (squares) and K40A RNASE4 (circles). Data shown are mean ± SEM of four independent preparations with triplicates in each enzyme concentration.

    Journal: Angiogenesis

    Article Title: Ribonuclease 4 protects neuron degeneration by promoting angiogenesis, neurogenesis, and neuronal survival under stress

    doi: 10.1007/s10456-012-9322-9

    Figure Lengend Snippet: Ribonucleolytic activity of recombinant wide type RNASE4 and the K40A variant. a SDS-PAGE and Coomassie blue staining of WT and K40A RNASE4 from one of the four preparations. Recombinant ANG protein was included as a control. b Ribonuclolysis of yeast tRNA by WT RNASE4 (squares) and K40A RNASE4 (circles). Data shown are mean ± SEM of four independent preparations with triplicates in each enzyme concentration.

    Article Snippet: Mouse Rnase4, forward, 5′- GGGTAATACGACTCACTATAGGGCGAtccgggtccaggcactttcta-3′; reverse, 5′-gtg ctggttcttgccctgtatcta-3′. cRNA probes were produced by in vitro transcription with T7 RNA polymerase from 1 μg of the above purified PCR products as template and were labeled with digoxigenin (Roche) per manufacturer’s protocol.

    Techniques: Activity Assay, Recombinant, Variant Assay, SDS Page, Staining, Concentration Assay

    RNASE4 induces endothelial cell tube formation. a Microscopic images of endothelial cell tubes formed in various concentration of WT and K40A RNASE4. ANG and PBS were used as positive and negative controls, respectively. The images shown are representative of at least four sections from three independent experiments. Bar, 0.1 mm. ( B ) Number of circled tubular structures per mm 2 . Values are mean ± SEM of three independent experiments. Statistical analysis was performed by two-way ANOVA. *, p

    Journal: Angiogenesis

    Article Title: Ribonuclease 4 protects neuron degeneration by promoting angiogenesis, neurogenesis, and neuronal survival under stress

    doi: 10.1007/s10456-012-9322-9

    Figure Lengend Snippet: RNASE4 induces endothelial cell tube formation. a Microscopic images of endothelial cell tubes formed in various concentration of WT and K40A RNASE4. ANG and PBS were used as positive and negative controls, respectively. The images shown are representative of at least four sections from three independent experiments. Bar, 0.1 mm. ( B ) Number of circled tubular structures per mm 2 . Values are mean ± SEM of three independent experiments. Statistical analysis was performed by two-way ANOVA. *, p

    Article Snippet: Mouse Rnase4, forward, 5′- GGGTAATACGACTCACTATAGGGCGAtccgggtccaggcactttcta-3′; reverse, 5′-gtg ctggttcttgccctgtatcta-3′. cRNA probes were produced by in vitro transcription with T7 RNA polymerase from 1 μg of the above purified PCR products as template and were labeled with digoxigenin (Roche) per manufacturer’s protocol.

    Techniques: Concentration Assay

    RNASE4 stimulates endothelial sprouts from mouse aortic explants. a Outward growth of endothelial sprouts from aortic rings that have been flipped inside out. The images are representative of three rings from one of three repeats. Bar, 0.1 mm. ANG and PBS were used as positive and negative controls, respectively. b ImageJ analysis of the area covered by endothelial sprouts from flipped aortic rings. Data are presented as mean ± SEM from three independent experiments. c Inward growth of endothelial sprouts from unflipped mouse aortic rings. The images are representative of three rings from one of two repeats. d ImageJ analysis the area covered by endothelial sprouts from unflipped aortic rings. Data are presented as mean ± SEM from two independent experiments. Bar, 1.0 mm. Statistical analysis was performed by two-way ANOVA. *, p

    Journal: Angiogenesis

    Article Title: Ribonuclease 4 protects neuron degeneration by promoting angiogenesis, neurogenesis, and neuronal survival under stress

    doi: 10.1007/s10456-012-9322-9

    Figure Lengend Snippet: RNASE4 stimulates endothelial sprouts from mouse aortic explants. a Outward growth of endothelial sprouts from aortic rings that have been flipped inside out. The images are representative of three rings from one of three repeats. Bar, 0.1 mm. ANG and PBS were used as positive and negative controls, respectively. b ImageJ analysis of the area covered by endothelial sprouts from flipped aortic rings. Data are presented as mean ± SEM from three independent experiments. c Inward growth of endothelial sprouts from unflipped mouse aortic rings. The images are representative of three rings from one of two repeats. d ImageJ analysis the area covered by endothelial sprouts from unflipped aortic rings. Data are presented as mean ± SEM from two independent experiments. Bar, 1.0 mm. Statistical analysis was performed by two-way ANOVA. *, p

    Article Snippet: Mouse Rnase4, forward, 5′- GGGTAATACGACTCACTATAGGGCGAtccgggtccaggcactttcta-3′; reverse, 5′-gtg ctggttcttgccctgtatcta-3′. cRNA probes were produced by in vitro transcription with T7 RNA polymerase from 1 μg of the above purified PCR products as template and were labeled with digoxigenin (Roche) per manufacturer’s protocol.

    Techniques:

    RNASE4 induces neovessel growth into Matrigel plug implanted under mouse skin. a IHC staining with vWF antibodies. The images shown are representative of at least four sections from three independent experiments. b ImageJ analysis of vWF-positive spots. Data are presented as mean ± SEM from three independent experiments. Bar, 100 μm. Statistical analysis was performed by two-way ANOVA. *, p

    Journal: Angiogenesis

    Article Title: Ribonuclease 4 protects neuron degeneration by promoting angiogenesis, neurogenesis, and neuronal survival under stress

    doi: 10.1007/s10456-012-9322-9

    Figure Lengend Snippet: RNASE4 induces neovessel growth into Matrigel plug implanted under mouse skin. a IHC staining with vWF antibodies. The images shown are representative of at least four sections from three independent experiments. b ImageJ analysis of vWF-positive spots. Data are presented as mean ± SEM from three independent experiments. Bar, 100 μm. Statistical analysis was performed by two-way ANOVA. *, p

    Article Snippet: Mouse Rnase4, forward, 5′- GGGTAATACGACTCACTATAGGGCGAtccgggtccaggcactttcta-3′; reverse, 5′-gtg ctggttcttgccctgtatcta-3′. cRNA probes were produced by in vitro transcription with T7 RNA polymerase from 1 μg of the above purified PCR products as template and were labeled with digoxigenin (Roche) per manufacturer’s protocol.

    Techniques: Immunohistochemistry, Staining

    Effect of RNASE4 on mouse embryonic cortical neurons. a Cortical neurons were isolated from E14 mouse embryos and cultured in neurobasal medium in the presence of 0.2 μg/ml RNASE4 or ANG for 12 days with a medium change on day 6. B27 was used as a positive control and BSA at 0.2 μg/ml was used as a negative control. The images are representative of at least four areas from three independent experiments. Bar, 0.5 mm. b ImageJ analysis of the length of the neurofilaments. Data shown are means ± SEM of three independent experiments in triplicates. Statistical analysis was performed by two-way ANOVA. **, p

    Journal: Angiogenesis

    Article Title: Ribonuclease 4 protects neuron degeneration by promoting angiogenesis, neurogenesis, and neuronal survival under stress

    doi: 10.1007/s10456-012-9322-9

    Figure Lengend Snippet: Effect of RNASE4 on mouse embryonic cortical neurons. a Cortical neurons were isolated from E14 mouse embryos and cultured in neurobasal medium in the presence of 0.2 μg/ml RNASE4 or ANG for 12 days with a medium change on day 6. B27 was used as a positive control and BSA at 0.2 μg/ml was used as a negative control. The images are representative of at least four areas from three independent experiments. Bar, 0.5 mm. b ImageJ analysis of the length of the neurofilaments. Data shown are means ± SEM of three independent experiments in triplicates. Statistical analysis was performed by two-way ANOVA. **, p

    Article Snippet: Mouse Rnase4, forward, 5′- GGGTAATACGACTCACTATAGGGCGAtccgggtccaggcactttcta-3′; reverse, 5′-gtg ctggttcttgccctgtatcta-3′. cRNA probes were produced by in vitro transcription with T7 RNA polymerase from 1 μg of the above purified PCR products as template and were labeled with digoxigenin (Roche) per manufacturer’s protocol.

    Techniques: Isolation, Cell Culture, Positive Control, Negative Control

    Ribonucleolytic activity of RNASE4 is essential for its angiogenic activity. a RNASE4 proteins was treated with 5 mM DEPC and its ribonucleolytic activity was examined by yeast tRNA assay. b DEPC-treated RNASE4 fails to cleave rRNA. c DEPC treatment abolishes angiogenic activity of RNASE4.

    Journal: Angiogenesis

    Article Title: Ribonuclease 4 protects neuron degeneration by promoting angiogenesis, neurogenesis, and neuronal survival under stress

    doi: 10.1007/s10456-012-9322-9

    Figure Lengend Snippet: Ribonucleolytic activity of RNASE4 is essential for its angiogenic activity. a RNASE4 proteins was treated with 5 mM DEPC and its ribonucleolytic activity was examined by yeast tRNA assay. b DEPC-treated RNASE4 fails to cleave rRNA. c DEPC treatment abolishes angiogenic activity of RNASE4.

    Article Snippet: Mouse Rnase4, forward, 5′- GGGTAATACGACTCACTATAGGGCGAtccgggtccaggcactttcta-3′; reverse, 5′-gtg ctggttcttgccctgtatcta-3′. cRNA probes were produced by in vitro transcription with T7 RNA polymerase from 1 μg of the above purified PCR products as template and were labeled with digoxigenin (Roche) per manufacturer’s protocol.

    Techniques: Activity Assay

    Related to Figure 2j. RNASEH2A P40D/Y210A is a separation-of-function mutant that cannot excise single DNA-embedded ribonucleotides, but cleaves RNA:DNA heteroduplexes (similar to the yeast rnh201-P45D-Y219A mutant 16 ). a, Schematic depicting enzymatic activity against two different RNase H2 substrates (DRD:DNA, dsDNA with embedded ribonucleotide, or RNA:DNA hybrids) in cell lines used in b-d and Fig 2j . WT and RNASEH2A-KO cells were transduced with either an empty vector (EV) or the indicated RNASEH2A constructs. b, Complementation of HeLa RNASEH2A-KO cells with FLAG-tagged RNASEH2A variants restores RNase H2 complex protein levels. WCEs from HeLa WT and RNASEH2A-KO cells stably expressing indicated lentiviral constructs were processed for immunoblotting with the indicated antibodies. Vinculin, loading control. Asterisk indicates a non-specific band. Representative of n = 3 biologically independent experiments. c , d, Complementation of HeLa RNASEH2A-KO cells with WT RNASEH2A, but not with the D34A/D169A (catalytic-dead) or P40D/Y210A (separation-of-function) mutants, rescues increased levels of genome-embedded ribonucleotides. c , Total nucleic acids from the cell lines shown in a , b were treated with recombinant RNase H2 and separated by alkaline agarose gel electrophoresis (representative of n = 4 experiments). d, Densitometric quantification of alkaline gel shown in c . e , Purified human RNase H2 complexes consisting of RNASEH2B, RNASEH2C and either RNASEH2A WT, P40D/Y210A or D34A/D169A subunits separated by SDS-PAGE and stained with Coomassie Blue ( n = 1). f-k , RNase H2 activity assays with fluorescein-labeled RNA:DNA substrate ( f ) or double-stranded DNA with a single incorporated ribonucleotide (DRD:DNA) ( g ) and increasing amounts of recombinant WT, P40D/Y210A or D34A/D169A RNase H2. Products were separated by polyacrylamide gel electrophoresis and detected by fluorescence imaging. Representative of n = 3 biologically independent experiments. h,k, Quantification of f , g . Product signal plotted relative to substrate signal per lane. Mean ±SD ( n = 3 biologically independent experiments).

    Journal: Nature

    Article Title: CRISPR screens identify genomic ribonucleotides as a source of PARP-trapping lesions

    doi: 10.1038/s41586-018-0291-z

    Figure Lengend Snippet: Related to Figure 2j. RNASEH2A P40D/Y210A is a separation-of-function mutant that cannot excise single DNA-embedded ribonucleotides, but cleaves RNA:DNA heteroduplexes (similar to the yeast rnh201-P45D-Y219A mutant 16 ). a, Schematic depicting enzymatic activity against two different RNase H2 substrates (DRD:DNA, dsDNA with embedded ribonucleotide, or RNA:DNA hybrids) in cell lines used in b-d and Fig 2j . WT and RNASEH2A-KO cells were transduced with either an empty vector (EV) or the indicated RNASEH2A constructs. b, Complementation of HeLa RNASEH2A-KO cells with FLAG-tagged RNASEH2A variants restores RNase H2 complex protein levels. WCEs from HeLa WT and RNASEH2A-KO cells stably expressing indicated lentiviral constructs were processed for immunoblotting with the indicated antibodies. Vinculin, loading control. Asterisk indicates a non-specific band. Representative of n = 3 biologically independent experiments. c , d, Complementation of HeLa RNASEH2A-KO cells with WT RNASEH2A, but not with the D34A/D169A (catalytic-dead) or P40D/Y210A (separation-of-function) mutants, rescues increased levels of genome-embedded ribonucleotides. c , Total nucleic acids from the cell lines shown in a , b were treated with recombinant RNase H2 and separated by alkaline agarose gel electrophoresis (representative of n = 4 experiments). d, Densitometric quantification of alkaline gel shown in c . e , Purified human RNase H2 complexes consisting of RNASEH2B, RNASEH2C and either RNASEH2A WT, P40D/Y210A or D34A/D169A subunits separated by SDS-PAGE and stained with Coomassie Blue ( n = 1). f-k , RNase H2 activity assays with fluorescein-labeled RNA:DNA substrate ( f ) or double-stranded DNA with a single incorporated ribonucleotide (DRD:DNA) ( g ) and increasing amounts of recombinant WT, P40D/Y210A or D34A/D169A RNase H2. Products were separated by polyacrylamide gel electrophoresis and detected by fluorescence imaging. Representative of n = 3 biologically independent experiments. h,k, Quantification of f , g . Product signal plotted relative to substrate signal per lane. Mean ±SD ( n = 3 biologically independent experiments).

    Article Snippet: For alkaline gel electrophoresis, 500 ng of total nucleic acids were incubated with 1 pmol of purified recombinant human RNase H2 and 0.25 μg of DNase-free RNase (Roche) for 30 min at 37°C in 100 µl reaction buffer (60 mM KCl, 50 mM Tris-HCl pH 8.0, 10 mM MgCl2 , 0.01% BSA, 0.01% Triton X-100).

    Techniques: Mutagenesis, Activity Assay, Transduction, Plasmid Preparation, Construct, Stable Transfection, Expressing, Recombinant, Agarose Gel Electrophoresis, Purification, SDS Page, Staining, Labeling, Polyacrylamide Gel Electrophoresis, Fluorescence, Imaging

    Related to Figure 4. a,b, Proliferating cells, and not quiescent cells, are the major population of viable cells in ex-vivo cultured primary CLL patient samples irrespective of treatment group. Quantification of absolute ( a ) and relative ( b ) quiescent and proliferating cell numbers as determined by FACS analysis of the primary CLL samples used in Fig 4b,c . ( RNASEH2B WT, n = 8 individual samples; monoallelic deletion, n = 4 individual samples; biallelic deletion, n = 9 individual samples). Mean ± SD ( n = 3 technical replicate). FACS gating strategy for stimulated peripheral blood lymphocytes (PBLs) from CLL patients is shown in Supplementary Fig 2 . c , RNase H2-deficient primary CLL cells have reduced survival when cultured with olaparib. Mean of individual samples ± s.e.m. ( n = 3 biologically independent CLL samples / group, each analyzed in technical triplicates). P -value, two-way ANOVA. d, Talazoparib selectively inhibits the growth of RNASEH2A-KO xenograft tumours. RNASEH2A-KO cells complemented either with empty vector (EV) or RNASEH2A-WT were injected subcutaneously into bilateral flanks of CD-1 nude mice. Mice were randomized to either vehicle or talazoparib (0.333 mg/kg) treatment groups (n = 8 animals / group) and tumour volumes were measured twice-weekly. Data plotted as mean ± s.e.m. P -values. two-way ANOVA under the null hypothesis that talazoparib does not supress the tumour growth.

    Journal: Nature

    Article Title: CRISPR screens identify genomic ribonucleotides as a source of PARP-trapping lesions

    doi: 10.1038/s41586-018-0291-z

    Figure Lengend Snippet: Related to Figure 4. a,b, Proliferating cells, and not quiescent cells, are the major population of viable cells in ex-vivo cultured primary CLL patient samples irrespective of treatment group. Quantification of absolute ( a ) and relative ( b ) quiescent and proliferating cell numbers as determined by FACS analysis of the primary CLL samples used in Fig 4b,c . ( RNASEH2B WT, n = 8 individual samples; monoallelic deletion, n = 4 individual samples; biallelic deletion, n = 9 individual samples). Mean ± SD ( n = 3 technical replicate). FACS gating strategy for stimulated peripheral blood lymphocytes (PBLs) from CLL patients is shown in Supplementary Fig 2 . c , RNase H2-deficient primary CLL cells have reduced survival when cultured with olaparib. Mean of individual samples ± s.e.m. ( n = 3 biologically independent CLL samples / group, each analyzed in technical triplicates). P -value, two-way ANOVA. d, Talazoparib selectively inhibits the growth of RNASEH2A-KO xenograft tumours. RNASEH2A-KO cells complemented either with empty vector (EV) or RNASEH2A-WT were injected subcutaneously into bilateral flanks of CD-1 nude mice. Mice were randomized to either vehicle or talazoparib (0.333 mg/kg) treatment groups (n = 8 animals / group) and tumour volumes were measured twice-weekly. Data plotted as mean ± s.e.m. P -values. two-way ANOVA under the null hypothesis that talazoparib does not supress the tumour growth.

    Article Snippet: For alkaline gel electrophoresis, 500 ng of total nucleic acids were incubated with 1 pmol of purified recombinant human RNase H2 and 0.25 μg of DNase-free RNase (Roche) for 30 min at 37°C in 100 µl reaction buffer (60 mM KCl, 50 mM Tris-HCl pH 8.0, 10 mM MgCl2 , 0.01% BSA, 0.01% Triton X-100).

    Techniques: Ex Vivo, Cell Culture, FACS, Plasmid Preparation, Injection, Mouse Assay

    Related to Figure 2. a-d , HR is not affected by inactivation of RNase H2. a, Representative micrographs ( n = 3 biologically independent experiments) of RPE1-hTERT Cas9 TP53-KO (WT) and RNASEH2A-KO cells exposed to 3 Gy of X-rays (IR) and processed for γ-H2AX and RAD51 immunofluorescence (IF) 4 h later. b. Quantification of the experiment in a at the indicated time points after IR, plotted as percentage of cells with > 5 γ-H2AX and RAD51 colocalizing foci. Individual values (open circles) with mean (red lines, n = 3 biologically independent experiments). P values, unpaired two-tailed t-test. c, Representative ( n = 3 biologically independent experiments) quantitative image-based cytometry (QIBC) plots of DR-GFP experiments in Fig 2e . Each point shows the mean GFP and RNASEH2A IF intensities per nucleus of mock- or I-SceI-transfected HeLa DR-GFP cells transduced with indicated Cas9/sgRNA constructs (EV = empty vector). Dashed lines separate RNASEH2A+/- and GFP+/- cell populations. d , Quantification of RNASEH2A+ cells in DR-GFP experiments shown in c and Fig 2e as determined by QIBC. Individual values (open circles) with mean (red lines; n = 3 biologically independent experiments). e-h, Replication-dependent endogenous DNA damage in RNase H2-deficient cells. e, Representative ( n = 3 biologically independent experiments) micrographs for experiments quantified in Fig 2g. γ-H2AX immunofluorescence (IF) in EdU positive (EdU+) and negative (EdU-) WT and RNASEH2A-KO HeLa cells. Scale bars, 5 µm. f , Quantification of γ-H2AX foci per nucleus in experiments shown in e and Fig 2g . Dots, foci number in individual nuclei. Red lines, mean ( n = 3 biologically independent experiments). g,h . HeLa WT and RNASEH2A-KO cells were treated with aphidicolin and EdU as indicated in the schematic (top), and immunostained with antibodies to γ - H2AX. Mean number of foci per EdU-positive (EdU+) nucleus in each experiment ( g , open circles) or the number of foci in individual EdU+ nuclei ( h , dots). Red lines, mean ( n = 3 biologically independent experiments, ≥100 cells / sample / experiment analyzed). P value, unpaired two-tailed t-test. i, j, Increased poly(ADP-ribosylation) of PARP1 in G1 as well as in S/G2/M phases in RNASEH2A-KO cells. i , Representative ( n = 2 biologically independent experiments) FACS plots of HeLa WT and RNASEH2A KO cells expressing the FUCCI cell cycle reporters mKO2-Cdt1 and mAG-Geminin 32 . j, PARP1 immunoprecipitates from WCEs of FUCCI-sorted G1 or S/G2/M HeLa WT and RNASEH2A-KO cells, probed with the indicated antibodies in immunoblotting (representative of n = 2 biologically independent experiments). Tubulin, loading control. Densitometric quantification of PAR signals normalized to immunoprecipitated PARP1 is shown as fold changes from WT to RNASEH2A-KO cells. k-o , Inactivation of RNase H2 in BRCA1- or BRCA2 -deficient backgrounds results in synthetic lethality. k, BRCA1 and BRCA2 expression, respectively, in RPE1-hTERT TP53-KO WT and BRCA1-KO (top) or DLD-1 WT and BRCA2-KO (bottom) cells. WCEs were processed for immunoblotting with the indicated antibodies. Tubulin and KAP1, loading controls. Representative of n ≥ 2 biologically independent experiments. l, RNase H2 levels in cells used in m, n, o (bottom) and Fig 2i . Cells were transduced with the indicated sgRNA- (top) or Cas9/sgRNA vectors (bottom; EV = empty vector) and processed for RNASEH2A IF. Each point represents mean RNASEH2A intensity per nucleus as measured by QIBC ( n = 1 experiment). ≥2000 cells analyzed per sample. Percentages of RNASEH2A+ cells in individual samples are shown above each plot. m, Representative images ( n = 3 biologically independent experiments) of clonogenic survival assays quantified in Fig 2i . n, o, Synthetic lethality after inactivation of RNASEH2A or RNASEH2B in BRCA2 -deficient cells. Clonogenic survival of DLD-1 WT and BRCA2-KO cells was assessed after transduction with indicated Cas9/sgRNA vectors. n , Representative images of n = 3 biologically independent experiments. o, Quantification of the experiment in n . Individual values (open circles) with mean (red lines; n = 3 biologically independent experiments). P values, unpaired two-tailed t-test.

    Journal: Nature

    Article Title: CRISPR screens identify genomic ribonucleotides as a source of PARP-trapping lesions

    doi: 10.1038/s41586-018-0291-z

    Figure Lengend Snippet: Related to Figure 2. a-d , HR is not affected by inactivation of RNase H2. a, Representative micrographs ( n = 3 biologically independent experiments) of RPE1-hTERT Cas9 TP53-KO (WT) and RNASEH2A-KO cells exposed to 3 Gy of X-rays (IR) and processed for γ-H2AX and RAD51 immunofluorescence (IF) 4 h later. b. Quantification of the experiment in a at the indicated time points after IR, plotted as percentage of cells with > 5 γ-H2AX and RAD51 colocalizing foci. Individual values (open circles) with mean (red lines, n = 3 biologically independent experiments). P values, unpaired two-tailed t-test. c, Representative ( n = 3 biologically independent experiments) quantitative image-based cytometry (QIBC) plots of DR-GFP experiments in Fig 2e . Each point shows the mean GFP and RNASEH2A IF intensities per nucleus of mock- or I-SceI-transfected HeLa DR-GFP cells transduced with indicated Cas9/sgRNA constructs (EV = empty vector). Dashed lines separate RNASEH2A+/- and GFP+/- cell populations. d , Quantification of RNASEH2A+ cells in DR-GFP experiments shown in c and Fig 2e as determined by QIBC. Individual values (open circles) with mean (red lines; n = 3 biologically independent experiments). e-h, Replication-dependent endogenous DNA damage in RNase H2-deficient cells. e, Representative ( n = 3 biologically independent experiments) micrographs for experiments quantified in Fig 2g. γ-H2AX immunofluorescence (IF) in EdU positive (EdU+) and negative (EdU-) WT and RNASEH2A-KO HeLa cells. Scale bars, 5 µm. f , Quantification of γ-H2AX foci per nucleus in experiments shown in e and Fig 2g . Dots, foci number in individual nuclei. Red lines, mean ( n = 3 biologically independent experiments). g,h . HeLa WT and RNASEH2A-KO cells were treated with aphidicolin and EdU as indicated in the schematic (top), and immunostained with antibodies to γ - H2AX. Mean number of foci per EdU-positive (EdU+) nucleus in each experiment ( g , open circles) or the number of foci in individual EdU+ nuclei ( h , dots). Red lines, mean ( n = 3 biologically independent experiments, ≥100 cells / sample / experiment analyzed). P value, unpaired two-tailed t-test. i, j, Increased poly(ADP-ribosylation) of PARP1 in G1 as well as in S/G2/M phases in RNASEH2A-KO cells. i , Representative ( n = 2 biologically independent experiments) FACS plots of HeLa WT and RNASEH2A KO cells expressing the FUCCI cell cycle reporters mKO2-Cdt1 and mAG-Geminin 32 . j, PARP1 immunoprecipitates from WCEs of FUCCI-sorted G1 or S/G2/M HeLa WT and RNASEH2A-KO cells, probed with the indicated antibodies in immunoblotting (representative of n = 2 biologically independent experiments). Tubulin, loading control. Densitometric quantification of PAR signals normalized to immunoprecipitated PARP1 is shown as fold changes from WT to RNASEH2A-KO cells. k-o , Inactivation of RNase H2 in BRCA1- or BRCA2 -deficient backgrounds results in synthetic lethality. k, BRCA1 and BRCA2 expression, respectively, in RPE1-hTERT TP53-KO WT and BRCA1-KO (top) or DLD-1 WT and BRCA2-KO (bottom) cells. WCEs were processed for immunoblotting with the indicated antibodies. Tubulin and KAP1, loading controls. Representative of n ≥ 2 biologically independent experiments. l, RNase H2 levels in cells used in m, n, o (bottom) and Fig 2i . Cells were transduced with the indicated sgRNA- (top) or Cas9/sgRNA vectors (bottom; EV = empty vector) and processed for RNASEH2A IF. Each point represents mean RNASEH2A intensity per nucleus as measured by QIBC ( n = 1 experiment). ≥2000 cells analyzed per sample. Percentages of RNASEH2A+ cells in individual samples are shown above each plot. m, Representative images ( n = 3 biologically independent experiments) of clonogenic survival assays quantified in Fig 2i . n, o, Synthetic lethality after inactivation of RNASEH2A or RNASEH2B in BRCA2 -deficient cells. Clonogenic survival of DLD-1 WT and BRCA2-KO cells was assessed after transduction with indicated Cas9/sgRNA vectors. n , Representative images of n = 3 biologically independent experiments. o, Quantification of the experiment in n . Individual values (open circles) with mean (red lines; n = 3 biologically independent experiments). P values, unpaired two-tailed t-test.

    Article Snippet: For alkaline gel electrophoresis, 500 ng of total nucleic acids were incubated with 1 pmol of purified recombinant human RNase H2 and 0.25 μg of DNase-free RNase (Roche) for 30 min at 37°C in 100 µl reaction buffer (60 mM KCl, 50 mM Tris-HCl pH 8.0, 10 mM MgCl2 , 0.01% BSA, 0.01% Triton X-100).

    Techniques: Immunofluorescence, Two Tailed Test, Cytometry, Transfection, Transduction, Construct, Plasmid Preparation, FACS, Expressing, Immunoprecipitation

    Related to Figure 2a,b. a, CRISPR-mediated inactivation of RNASEH2A or RNASEH2B in the cell lines used in this manuscript. WCEs of indicated cell lines and genotypes were processed for immunoblotting using antibodies against RNASEH2A, RNASEH2B or RNASEH2C. Vinculin, tubulin and GAPDH, loading controls. Representative immunoblots (of n ≥ 2 biologically independent experiments). b-d. Abolished RNase H2 enzymatic activity and increased levels of genome-embedded ribonucleotides in RNASEH2A-KO cells. b. Representative ( n = 3 biologically independent experiments) analysis of total nucleic acids from WT and RNASEH2A-KO HeLa cells treated with recombinant RNase H2 and separated by alkaline agarose gel electrophoresis. Ribonucleotide-containing genomic DNA from RNASEH2A-KO HeLa cells is nicked and therefore has increased electrophoretic mobility 13 . c, Densitometric quantification of the alkaline gel shown in b . d, Cleavage of an RNase H2-specific double-stranded DNA oligonucleotide with a single incorporated ribonucleotide (DRD:DNA; ribonucleotide position is shown in red) by WT and RNASEH2A-KO WCEs of the indicated cell types was measured using a fluorescence quenching-based assay 31 . Individual values (open circles) with mean (red lines, n = 3 biologically independent experiments). e-l, RNase H2 deficiency leads to PARPi sensitivity in multiple cell types. e-h, Clonogenic survival assays of the indicated cell lines treated with the indicated PARPi. Mean ±SD, normalized to untreated cells ( n = 3 biologically independent experiments). Solid lines, nonlinear least squares fit of the data to a three-parameter dose response model. h. EC50 values for olaparib (left) and talazoparib (right) in the indicated cell lines as determined by nonlinear least squares fitting of data in e, f, g and Fig 2a,b. Bars, EC50 value ± 95% confidence interval. i-l, Increased apoptosis in HeLa RNASEH2A-KO, SUM149PT Cas9 RNASEH2B-KO and HCT116 RNASEH2A-KO cells following PARPi treatment. i , Representative ( n = 3 biologically independent experiments) cleaved caspase-3 immunofluorescence / flow cytometry (IF/FACS) profiles of untreated and talazoparib-treated HeLa WT and RNASEH2A-KO cells. FSC = forward scatter. j-l, Percentages of cleaved caspase-3-positive (caspase-3+) cells of the indicated genotypes treated with the indicated PARPi. Individual values (coloured symbols) with mean (solid lines, n = 3 biologically independent experiments). Inset: Levels of cleaved caspase-3+ cells without PARPi treatment. Red lines, mean ( n = 3 biologically independent experiments). P values, unpaired two-tailed t-test. In a , d , g and l , HCT116 RNASEH2A-KO cells were transduced either with an empty vector (+EV) or a full-length RNASEH2A expression construct (+WT), where indicated.

    Journal: Nature

    Article Title: CRISPR screens identify genomic ribonucleotides as a source of PARP-trapping lesions

    doi: 10.1038/s41586-018-0291-z

    Figure Lengend Snippet: Related to Figure 2a,b. a, CRISPR-mediated inactivation of RNASEH2A or RNASEH2B in the cell lines used in this manuscript. WCEs of indicated cell lines and genotypes were processed for immunoblotting using antibodies against RNASEH2A, RNASEH2B or RNASEH2C. Vinculin, tubulin and GAPDH, loading controls. Representative immunoblots (of n ≥ 2 biologically independent experiments). b-d. Abolished RNase H2 enzymatic activity and increased levels of genome-embedded ribonucleotides in RNASEH2A-KO cells. b. Representative ( n = 3 biologically independent experiments) analysis of total nucleic acids from WT and RNASEH2A-KO HeLa cells treated with recombinant RNase H2 and separated by alkaline agarose gel electrophoresis. Ribonucleotide-containing genomic DNA from RNASEH2A-KO HeLa cells is nicked and therefore has increased electrophoretic mobility 13 . c, Densitometric quantification of the alkaline gel shown in b . d, Cleavage of an RNase H2-specific double-stranded DNA oligonucleotide with a single incorporated ribonucleotide (DRD:DNA; ribonucleotide position is shown in red) by WT and RNASEH2A-KO WCEs of the indicated cell types was measured using a fluorescence quenching-based assay 31 . Individual values (open circles) with mean (red lines, n = 3 biologically independent experiments). e-l, RNase H2 deficiency leads to PARPi sensitivity in multiple cell types. e-h, Clonogenic survival assays of the indicated cell lines treated with the indicated PARPi. Mean ±SD, normalized to untreated cells ( n = 3 biologically independent experiments). Solid lines, nonlinear least squares fit of the data to a three-parameter dose response model. h. EC50 values for olaparib (left) and talazoparib (right) in the indicated cell lines as determined by nonlinear least squares fitting of data in e, f, g and Fig 2a,b. Bars, EC50 value ± 95% confidence interval. i-l, Increased apoptosis in HeLa RNASEH2A-KO, SUM149PT Cas9 RNASEH2B-KO and HCT116 RNASEH2A-KO cells following PARPi treatment. i , Representative ( n = 3 biologically independent experiments) cleaved caspase-3 immunofluorescence / flow cytometry (IF/FACS) profiles of untreated and talazoparib-treated HeLa WT and RNASEH2A-KO cells. FSC = forward scatter. j-l, Percentages of cleaved caspase-3-positive (caspase-3+) cells of the indicated genotypes treated with the indicated PARPi. Individual values (coloured symbols) with mean (solid lines, n = 3 biologically independent experiments). Inset: Levels of cleaved caspase-3+ cells without PARPi treatment. Red lines, mean ( n = 3 biologically independent experiments). P values, unpaired two-tailed t-test. In a , d , g and l , HCT116 RNASEH2A-KO cells were transduced either with an empty vector (+EV) or a full-length RNASEH2A expression construct (+WT), where indicated.

    Article Snippet: For alkaline gel electrophoresis, 500 ng of total nucleic acids were incubated with 1 pmol of purified recombinant human RNase H2 and 0.25 μg of DNase-free RNase (Roche) for 30 min at 37°C in 100 µl reaction buffer (60 mM KCl, 50 mM Tris-HCl pH 8.0, 10 mM MgCl2 , 0.01% BSA, 0.01% Triton X-100).

    Techniques: CRISPR, Western Blot, Activity Assay, Recombinant, Agarose Gel Electrophoresis, Fluorescence, Immunofluorescence, Flow Cytometry, Cytometry, FACS, Two Tailed Test, Plasmid Preparation, Expressing, Construct

    Defective ribonucleotide excision repair causes PARPi sensitivity, DNA damage and synthetic lethality with BRCA1 deficiency. a,b, Reduced survival of HeLa RNASEH2A-KO cells after treatment with indicated PARPi. Mean ±SD, normalized to untreated cells. Solid lines, nonlinear least-squares fit to a three-parameter dose-response model. c-f, RNASEH2A-KO cells are HR-proficient. c,d, Normal RAD51 focus formation in RNASEH2A-KO HeLa cells after X-ray exposure. c, Representative micrographs of HeLa WT and RNASEH2A-KO cells stained with indicated antibodies ( n = 3 biologically independent experiments). Scale bar, 10 μm. d, Quantification. Percentage of cells with > 5 RAD51/γ-H2AX colocalizing foci at indicated time points. e, HR is not impaired in RNase H2-null cells. Quantification of gene conversion in DR-GFP reporter cells 11 transduced with Cas9 + sg RNASEH2A/B or empty vector (EV) ± I-SceI transfection. Values normalized to transfection efficiency of control GFP vector. f, Increased sister chromatid exchanges (SCEs) in RNASEH2A-KO cells. Representative micrographs of SCEs in WT and RNASEH2A-KO metaphases. Below, numbers of SCEs / chromosome (mean ±SD, n = 3 biologically independent experiments). Scale bars, 10 μm. g,h, Spontaneous replication-associated damage and increased PARP1 activation in RNASEH2A-KO cells. g, Quantification of mean γ-H2AX immunofluorescent foci number / nucleus in EdU positive (+) and -negative (-) WT and RNASEH2A-KO cells. h, Representative poly(ADP-ribose) (PAR) immunoblot of PARP1 immunoprecipitates (IP) from whole cell extracts (WCE). Mean fold-increase in PARylation between WT and RNASEH2A-KO indicated ( n = 3 biologically independent experiments, normalized to immunoprecipitated PARP1 levels). Tubulin and IgG heavy chain, loading controls. i , Synthetic lethality in combined absence of RNase H2 and BRCA1. Quantification of colony formation of BRCA1 -proficient (WT) and BRCA1-KO RPE1-hTERT Cas9 TP53-KO cells transduced with sg LacZ or sg RNASEH2B constructs. Open circles, individual values normalized to sg LacZ ; red lines, mean ( n = 3 biologically independent experiments). j , PARPi sensitivity is associated with ribonuclease excision repair (RER) deficiency. Survival of olaparib-treated HeLa WT and RNASEH2A-KO cells transduced with indicated FLAG-tagged constructs. Mean ±SD, normalized to untreated cells ( n = 3 biologically independent experiments). Solid lines, nonlinear least squares fit to a three-parameter dose response model. For d , e , and g : open circles, individual values; red lines, mean [ n = 3 biologically independent experiments; ≥100 ( d, g ) and ≥1000 ( e ) cells / sample / experiment analyzed]. P values in d-g and i , unpaired two-tailed t-test. See also ED Fig 2 - 4 .

    Journal: Nature

    Article Title: CRISPR screens identify genomic ribonucleotides as a source of PARP-trapping lesions

    doi: 10.1038/s41586-018-0291-z

    Figure Lengend Snippet: Defective ribonucleotide excision repair causes PARPi sensitivity, DNA damage and synthetic lethality with BRCA1 deficiency. a,b, Reduced survival of HeLa RNASEH2A-KO cells after treatment with indicated PARPi. Mean ±SD, normalized to untreated cells. Solid lines, nonlinear least-squares fit to a three-parameter dose-response model. c-f, RNASEH2A-KO cells are HR-proficient. c,d, Normal RAD51 focus formation in RNASEH2A-KO HeLa cells after X-ray exposure. c, Representative micrographs of HeLa WT and RNASEH2A-KO cells stained with indicated antibodies ( n = 3 biologically independent experiments). Scale bar, 10 μm. d, Quantification. Percentage of cells with > 5 RAD51/γ-H2AX colocalizing foci at indicated time points. e, HR is not impaired in RNase H2-null cells. Quantification of gene conversion in DR-GFP reporter cells 11 transduced with Cas9 + sg RNASEH2A/B or empty vector (EV) ± I-SceI transfection. Values normalized to transfection efficiency of control GFP vector. f, Increased sister chromatid exchanges (SCEs) in RNASEH2A-KO cells. Representative micrographs of SCEs in WT and RNASEH2A-KO metaphases. Below, numbers of SCEs / chromosome (mean ±SD, n = 3 biologically independent experiments). Scale bars, 10 μm. g,h, Spontaneous replication-associated damage and increased PARP1 activation in RNASEH2A-KO cells. g, Quantification of mean γ-H2AX immunofluorescent foci number / nucleus in EdU positive (+) and -negative (-) WT and RNASEH2A-KO cells. h, Representative poly(ADP-ribose) (PAR) immunoblot of PARP1 immunoprecipitates (IP) from whole cell extracts (WCE). Mean fold-increase in PARylation between WT and RNASEH2A-KO indicated ( n = 3 biologically independent experiments, normalized to immunoprecipitated PARP1 levels). Tubulin and IgG heavy chain, loading controls. i , Synthetic lethality in combined absence of RNase H2 and BRCA1. Quantification of colony formation of BRCA1 -proficient (WT) and BRCA1-KO RPE1-hTERT Cas9 TP53-KO cells transduced with sg LacZ or sg RNASEH2B constructs. Open circles, individual values normalized to sg LacZ ; red lines, mean ( n = 3 biologically independent experiments). j , PARPi sensitivity is associated with ribonuclease excision repair (RER) deficiency. Survival of olaparib-treated HeLa WT and RNASEH2A-KO cells transduced with indicated FLAG-tagged constructs. Mean ±SD, normalized to untreated cells ( n = 3 biologically independent experiments). Solid lines, nonlinear least squares fit to a three-parameter dose response model. For d , e , and g : open circles, individual values; red lines, mean [ n = 3 biologically independent experiments; ≥100 ( d, g ) and ≥1000 ( e ) cells / sample / experiment analyzed]. P values in d-g and i , unpaired two-tailed t-test. See also ED Fig 2 - 4 .

    Article Snippet: For alkaline gel electrophoresis, 500 ng of total nucleic acids were incubated with 1 pmol of purified recombinant human RNase H2 and 0.25 μg of DNase-free RNase (Roche) for 30 min at 37°C in 100 µl reaction buffer (60 mM KCl, 50 mM Tris-HCl pH 8.0, 10 mM MgCl2 , 0.01% BSA, 0.01% Triton X-100).

    Techniques: Staining, Transduction, Plasmid Preparation, Transfection, Activation Assay, Immunoprecipitation, Construct, Two Tailed Test

    RNase H2-deficient cells are more sensitive to PARPi than DNA polymerase β mutants. a, Schematic representation of the POLBΔ188-190 CRISPR mutation. The Mg 2+ -coordinating aspartate residues (D190, D192 and D256, red triangles) are highlighted in the domain structure of the human Polβ protein. The sgRNA target site and antibody epitope are indicated by black lines. b, WCEs from parental RPE1-hTERT Cas9 TP53-KO cells and two POLBΔ188-190 clones were processed for immunoblotting with antibodies to Polβ and tubulin (loading control). Representative of n = 2 biologically independent experiments. c, The POLBΔ188-190 mutation impairs base excision repair. RPE1-hTERT Cas9 TP53-KO WT or POLBΔ188-190 cells were exposed to different concentrations of methyl-methanesulfonate (MMS) for 24 h, followed by growth in drug-free media for an additional 48 h. Cell viability was determined by the Cell Titer Glo assay. d, Sensitivity of RPE1-hTERT Cas9 TP53-KO WT, RNASEH2A-KO and POLBΔ188-190 cells to indicated talazoparib concentrations in clonogenic survival assays. Data in c and d represent mean ±SD, normalized to untreated cells ( n = 3 biologically independent experiments). Solid lines denote a nonlinear least-squares fit to a three-parameter dose response model.

    Journal: Nature

    Article Title: CRISPR screens identify genomic ribonucleotides as a source of PARP-trapping lesions

    doi: 10.1038/s41586-018-0291-z

    Figure Lengend Snippet: RNase H2-deficient cells are more sensitive to PARPi than DNA polymerase β mutants. a, Schematic representation of the POLBΔ188-190 CRISPR mutation. The Mg 2+ -coordinating aspartate residues (D190, D192 and D256, red triangles) are highlighted in the domain structure of the human Polβ protein. The sgRNA target site and antibody epitope are indicated by black lines. b, WCEs from parental RPE1-hTERT Cas9 TP53-KO cells and two POLBΔ188-190 clones were processed for immunoblotting with antibodies to Polβ and tubulin (loading control). Representative of n = 2 biologically independent experiments. c, The POLBΔ188-190 mutation impairs base excision repair. RPE1-hTERT Cas9 TP53-KO WT or POLBΔ188-190 cells were exposed to different concentrations of methyl-methanesulfonate (MMS) for 24 h, followed by growth in drug-free media for an additional 48 h. Cell viability was determined by the Cell Titer Glo assay. d, Sensitivity of RPE1-hTERT Cas9 TP53-KO WT, RNASEH2A-KO and POLBΔ188-190 cells to indicated talazoparib concentrations in clonogenic survival assays. Data in c and d represent mean ±SD, normalized to untreated cells ( n = 3 biologically independent experiments). Solid lines denote a nonlinear least-squares fit to a three-parameter dose response model.

    Article Snippet: For alkaline gel electrophoresis, 500 ng of total nucleic acids were incubated with 1 pmol of purified recombinant human RNase H2 and 0.25 μg of DNase-free RNase (Roche) for 30 min at 37°C in 100 µl reaction buffer (60 mM KCl, 50 mM Tris-HCl pH 8.0, 10 mM MgCl2 , 0.01% BSA, 0.01% Triton X-100).

    Techniques: CRISPR, Mutagenesis, Clone Assay, Glo Assay

    Talazoparib selectively suppresses growth of RNase H2 deficient tumours. a-c, PARP inhibitors selectively kill RNASEH2B- deficient chronic lymphocytic leukemia (CLL) primary cancer cells. a, RNASEH2B deletion frequency in a panel of 100 primary CLL samples, determined by multiplex ligation-dependent probe amplification (MLPA). b, Reduced RNase H2 activity in lysates from CLL samples with monoallelic and biallelic RNASEH2B deletions. Top, substrate schematic. Individual data points, mean of technical duplicates for each sample. Red lines, mean of individual genotypes ( n = 8 WT, 4 monoallelic and 9 biallelic deleted biologically independent primary CLL samples). Data normalized to mean of RNASEH2B-WT samples. c , Reduced survival of CLL cells with monoallelic and biallelic RNASEH2B loss following treatment with talazoparib. Individual points, mean ± s.e.m. ( n = 8, 4 and 9 CLL samples as in b ), each analysed in technical triplicates. P -values, unpaired two-tailed t-test ( b ) and two-way ANOVA ( c ). d , Selective inhibition of RNASEH2A-KO xenograft tumour growth. HCT116 TP53-KO RNASEH2A-WT or -KO cells were injected subcutaneously into bilateral flanks of CD-1 nude mice. Mice were randomized to either vehicle or talazoparib (0.333 mg/kg) treatment groups ( n = 8 animals / group) and tumour volumes measured twice-weekly. Mean ± s.e.m. P -value, two-way ANOVA. e , Model. Genome-embedded ribonucleotides (R) can be processed by TOP1 as an alternative to RNase H2-dependent RER. DNA lesions that engage PARP1 (black circles) are formed as a result, and PARP inhibitors induce PARP1 trapping on these TOP1-dependent lesions, causing replication arrest, persistent DNA damage and cell death. See also ED Fig 7 , 8 , ED Table 1 and Supplementary Table 3 .

    Journal: Nature

    Article Title: CRISPR screens identify genomic ribonucleotides as a source of PARP-trapping lesions

    doi: 10.1038/s41586-018-0291-z

    Figure Lengend Snippet: Talazoparib selectively suppresses growth of RNase H2 deficient tumours. a-c, PARP inhibitors selectively kill RNASEH2B- deficient chronic lymphocytic leukemia (CLL) primary cancer cells. a, RNASEH2B deletion frequency in a panel of 100 primary CLL samples, determined by multiplex ligation-dependent probe amplification (MLPA). b, Reduced RNase H2 activity in lysates from CLL samples with monoallelic and biallelic RNASEH2B deletions. Top, substrate schematic. Individual data points, mean of technical duplicates for each sample. Red lines, mean of individual genotypes ( n = 8 WT, 4 monoallelic and 9 biallelic deleted biologically independent primary CLL samples). Data normalized to mean of RNASEH2B-WT samples. c , Reduced survival of CLL cells with monoallelic and biallelic RNASEH2B loss following treatment with talazoparib. Individual points, mean ± s.e.m. ( n = 8, 4 and 9 CLL samples as in b ), each analysed in technical triplicates. P -values, unpaired two-tailed t-test ( b ) and two-way ANOVA ( c ). d , Selective inhibition of RNASEH2A-KO xenograft tumour growth. HCT116 TP53-KO RNASEH2A-WT or -KO cells were injected subcutaneously into bilateral flanks of CD-1 nude mice. Mice were randomized to either vehicle or talazoparib (0.333 mg/kg) treatment groups ( n = 8 animals / group) and tumour volumes measured twice-weekly. Mean ± s.e.m. P -value, two-way ANOVA. e , Model. Genome-embedded ribonucleotides (R) can be processed by TOP1 as an alternative to RNase H2-dependent RER. DNA lesions that engage PARP1 (black circles) are formed as a result, and PARP inhibitors induce PARP1 trapping on these TOP1-dependent lesions, causing replication arrest, persistent DNA damage and cell death. See also ED Fig 7 , 8 , ED Table 1 and Supplementary Table 3 .

    Article Snippet: For alkaline gel electrophoresis, 500 ng of total nucleic acids were incubated with 1 pmol of purified recombinant human RNase H2 and 0.25 μg of DNase-free RNase (Roche) for 30 min at 37°C in 100 µl reaction buffer (60 mM KCl, 50 mM Tris-HCl pH 8.0, 10 mM MgCl2 , 0.01% BSA, 0.01% Triton X-100).

    Techniques: Multiplex Assay, Ligation, Amplification, Multiplex Ligation-dependent Probe Amplification, Activity Assay, Two Tailed Test, Inhibition, Injection, Mouse Assay

    Related to Fig 3a-c. PARP1 trapping is the underlying cause of PARPi sensitivity in RNase H2-deficient cells. a, Schematic representation of CRISPR screens for suppressors of talazoparib sensitivity in RNase H2-deficient cells. Cas9-expressing cells were transduced with the TKOv1 library, talazoparib was added on day 6 (t6; HeLa: 20 nM, RPE1-hTERT: 50 nM) and cells were cultured in its presence until day 18 (t18). Cells were subcultured once at day 12 (RPE1) or 13 (HeLa). sgRNA representations in the initial (t6) and final (t18) populations were quantified by next-generation sequencing. Gene knockouts that were enriched at t18 over t6 were identified by MAGeCK 33 . b, CRISPR-mediated inactivation of RNASEH2A and/or PARP1 in cell lines used in c-e and Fig 3b . WCEs were processed for immunoblotting with the indicated antibodies. KAP1, loading control. Representative of n = 2 biologically independent experiments. c-e, Loss of PARP1 restores PARPi-resistance in RNASEH2A-KO cells. c, Percentage of cleaved caspase-3+ HeLa cells of indicated genotypes with or without olaparib treatment measured by flow cytometry (FACS). Individual values (open circles) with mean (red lines, n = 3 biologically independent experiments; P -value, unpaired two-tailed t-test). d,e. Clonogenic survival assays with HeLa ( d ) and RPE1-hTERT ( e ) cells of the indicated genotypes treated with olaparib (left) or talazoparib (right). Mean ±SD ( n = 3 biologically independent experiments). Solid lines, nonlinear least squares fit to a three-parameter dose response model. f. Trapping activity of PARPi correlates with the ability to induce apoptosis in RNASEH2A-KO cells. Quantification of cleaved caspase-3-positive HeLa WT and RNASEH2B-KO cells without treatment or treated with the indicated PARPi. Individual values with mean (black lines, n = 3 biologically independent experiments). Note that PARP-trapping activity decreases as follows: talazoparib > olaparib > veliparib 4 , 17 . g , PARPi-induced S-phase arrest in RNASEH2A-KO cells is alleviated in the absence of PARP1. Top, schematic of talazoparib and EdU treatment. Bottom, representative ( n = 3 biologically independent experiments) EdU (pseudocolor plots) and DNA content (histograms) FACS profiles of untreated and talazoparib-treated HeLa WT, PARP1-KO , RNASEH2A-KO and PARP1-KO/RNASEH2A-KO cells. DNA content was determined by propidium iodide (PI) staining. h Quantification of mean γ-H2AX intensities in experiments shown in Fig 3c . Individual values (open circles) with mean (red lines, n = 3 biologically independent experiments, ≥10,000 cells / sample / experiment analyzed).

    Journal: Nature

    Article Title: CRISPR screens identify genomic ribonucleotides as a source of PARP-trapping lesions

    doi: 10.1038/s41586-018-0291-z

    Figure Lengend Snippet: Related to Fig 3a-c. PARP1 trapping is the underlying cause of PARPi sensitivity in RNase H2-deficient cells. a, Schematic representation of CRISPR screens for suppressors of talazoparib sensitivity in RNase H2-deficient cells. Cas9-expressing cells were transduced with the TKOv1 library, talazoparib was added on day 6 (t6; HeLa: 20 nM, RPE1-hTERT: 50 nM) and cells were cultured in its presence until day 18 (t18). Cells were subcultured once at day 12 (RPE1) or 13 (HeLa). sgRNA representations in the initial (t6) and final (t18) populations were quantified by next-generation sequencing. Gene knockouts that were enriched at t18 over t6 were identified by MAGeCK 33 . b, CRISPR-mediated inactivation of RNASEH2A and/or PARP1 in cell lines used in c-e and Fig 3b . WCEs were processed for immunoblotting with the indicated antibodies. KAP1, loading control. Representative of n = 2 biologically independent experiments. c-e, Loss of PARP1 restores PARPi-resistance in RNASEH2A-KO cells. c, Percentage of cleaved caspase-3+ HeLa cells of indicated genotypes with or without olaparib treatment measured by flow cytometry (FACS). Individual values (open circles) with mean (red lines, n = 3 biologically independent experiments; P -value, unpaired two-tailed t-test). d,e. Clonogenic survival assays with HeLa ( d ) and RPE1-hTERT ( e ) cells of the indicated genotypes treated with olaparib (left) or talazoparib (right). Mean ±SD ( n = 3 biologically independent experiments). Solid lines, nonlinear least squares fit to a three-parameter dose response model. f. Trapping activity of PARPi correlates with the ability to induce apoptosis in RNASEH2A-KO cells. Quantification of cleaved caspase-3-positive HeLa WT and RNASEH2B-KO cells without treatment or treated with the indicated PARPi. Individual values with mean (black lines, n = 3 biologically independent experiments). Note that PARP-trapping activity decreases as follows: talazoparib > olaparib > veliparib 4 , 17 . g , PARPi-induced S-phase arrest in RNASEH2A-KO cells is alleviated in the absence of PARP1. Top, schematic of talazoparib and EdU treatment. Bottom, representative ( n = 3 biologically independent experiments) EdU (pseudocolor plots) and DNA content (histograms) FACS profiles of untreated and talazoparib-treated HeLa WT, PARP1-KO , RNASEH2A-KO and PARP1-KO/RNASEH2A-KO cells. DNA content was determined by propidium iodide (PI) staining. h Quantification of mean γ-H2AX intensities in experiments shown in Fig 3c . Individual values (open circles) with mean (red lines, n = 3 biologically independent experiments, ≥10,000 cells / sample / experiment analyzed).

    Article Snippet: For alkaline gel electrophoresis, 500 ng of total nucleic acids were incubated with 1 pmol of purified recombinant human RNase H2 and 0.25 μg of DNase-free RNase (Roche) for 30 min at 37°C in 100 µl reaction buffer (60 mM KCl, 50 mM Tris-HCl pH 8.0, 10 mM MgCl2 , 0.01% BSA, 0.01% Triton X-100).

    Techniques: CRISPR, Expressing, Transduction, Cell Culture, Next-Generation Sequencing, Flow Cytometry, Cytometry, FACS, Two Tailed Test, Activity Assay, Staining

    Expression of RNase L blocks L1 RNP formation. HeLa-M cells were co-transfected with pES2TE1 and either an empty vector (pcDNA 3.0) or a plasmid that encodes an amino-terminal Myc-tagged RNase L expression plasmid. Immunofluorescent confocal microscopy was used to examine L1 ORF2p accumulation in cytoplasmic foci by exploiting the FLAG-HA epitope-tag in pES2TE1 48 h after transfection. The top labels indicate the antibodies used to detect the indicated proteins: anti-HA-ORF2p, red; anti-EBNA-1, green; anti-Myc RNase L, magenta. The labels on the left side of the figure indicate the empty vector or RNase L constructs that were co-transfected into cells. The rightmost column indicates the merged overlay staining. L1 ORF2p formed discrete cytoplasmic punctate localization in co-transfection experiments performed with the empty vector and RNase L catalytically inactive mutant (R667A), but not with WT RNase L. For each condition, either two or three slides were examined per experiment. About 200 cells were examined per slide and representative images were captured. The experiment was conducted three times (biological replicates) with similar results.

    Journal: Nucleic Acids Research

    Article Title: RNase L restricts the mobility of engineered retrotransposons in cultured human cells

    doi: 10.1093/nar/gkt1308

    Figure Lengend Snippet: Expression of RNase L blocks L1 RNP formation. HeLa-M cells were co-transfected with pES2TE1 and either an empty vector (pcDNA 3.0) or a plasmid that encodes an amino-terminal Myc-tagged RNase L expression plasmid. Immunofluorescent confocal microscopy was used to examine L1 ORF2p accumulation in cytoplasmic foci by exploiting the FLAG-HA epitope-tag in pES2TE1 48 h after transfection. The top labels indicate the antibodies used to detect the indicated proteins: anti-HA-ORF2p, red; anti-EBNA-1, green; anti-Myc RNase L, magenta. The labels on the left side of the figure indicate the empty vector or RNase L constructs that were co-transfected into cells. The rightmost column indicates the merged overlay staining. L1 ORF2p formed discrete cytoplasmic punctate localization in co-transfection experiments performed with the empty vector and RNase L catalytically inactive mutant (R667A), but not with WT RNase L. For each condition, either two or three slides were examined per experiment. About 200 cells were examined per slide and representative images were captured. The experiment was conducted three times (biological replicates) with similar results.

    Article Snippet: For enhanced green fluorescent protein (EGFP)-based retrotransposition assays, HeLa-M cells were transfected with 0.5 µg of an active (pLRE3-mEGFPI ) or inactive (pJM111-LRE3-mEGFPI ) L1 expression plasmid and 0.5 µg of a corresponding RNase L expression plasmid, using 3 µl of Fugene6 transfection reagent (Roche) per well.

    Techniques: Expressing, Transfection, Plasmid Preparation, Confocal Microscopy, Construct, Staining, Cotransfection, Mutagenesis

    Human RNase L alone does not affect G418-resistant foci formation. ( A ) Results from the Assay: HeLa-M cells were co-transfected with pcDNA 3.0 (Gibco/Life Technologies/InVitrogen) and either an empty vector (pFLAG-CMV-2) or an amino-terminal FLAG-tagged RNase L expression plasmid. The cells were subjected to selection for 10 days and G418-resistant foci were fixed and stained with crystal violet for visualization purposes. A representative tissue culture dish for each condition is shown. ( B ) Quantitation of the Assays: The X-axis depicts construct names. The Y-axis depicts the number of G418-resistant foci per cell culture dish. Quantification was performed as outlined in the legend to Figure 2 B. Data are shown as the mean ± standard deviation (SD) from a single experiment with three technical replicates. The experiment was conducted three times (biological replicates) with similar results. No statistically significant difference was found with one-way ANOVA and post hoc tests. ( C ) Protein expression analyses: The WT RNase L, catalytically inactive RNase L mutant (R667A) and constitutively active (NΔ385) RNase L mutant were detected from total cell lysates in western blots with anti-RNase L antibody 2 days after transfection. β-Actin served as loading and transfer control. Size standards are indicated in kDa at the left of the gel.

    Journal: Nucleic Acids Research

    Article Title: RNase L restricts the mobility of engineered retrotransposons in cultured human cells

    doi: 10.1093/nar/gkt1308

    Figure Lengend Snippet: Human RNase L alone does not affect G418-resistant foci formation. ( A ) Results from the Assay: HeLa-M cells were co-transfected with pcDNA 3.0 (Gibco/Life Technologies/InVitrogen) and either an empty vector (pFLAG-CMV-2) or an amino-terminal FLAG-tagged RNase L expression plasmid. The cells were subjected to selection for 10 days and G418-resistant foci were fixed and stained with crystal violet for visualization purposes. A representative tissue culture dish for each condition is shown. ( B ) Quantitation of the Assays: The X-axis depicts construct names. The Y-axis depicts the number of G418-resistant foci per cell culture dish. Quantification was performed as outlined in the legend to Figure 2 B. Data are shown as the mean ± standard deviation (SD) from a single experiment with three technical replicates. The experiment was conducted three times (biological replicates) with similar results. No statistically significant difference was found with one-way ANOVA and post hoc tests. ( C ) Protein expression analyses: The WT RNase L, catalytically inactive RNase L mutant (R667A) and constitutively active (NΔ385) RNase L mutant were detected from total cell lysates in western blots with anti-RNase L antibody 2 days after transfection. β-Actin served as loading and transfer control. Size standards are indicated in kDa at the left of the gel.

    Article Snippet: For enhanced green fluorescent protein (EGFP)-based retrotransposition assays, HeLa-M cells were transfected with 0.5 µg of an active (pLRE3-mEGFPI ) or inactive (pJM111-LRE3-mEGFPI ) L1 expression plasmid and 0.5 µg of a corresponding RNase L expression plasmid, using 3 µl of Fugene6 transfection reagent (Roche) per well.

    Techniques: Transfection, Plasmid Preparation, Expressing, Selection, Staining, Quantitation Assay, Construct, Cell Culture, Standard Deviation, Mutagenesis, Western Blot

    Expression of RNase L reduces L1 protein expression. ( A ) L1 protein expression: HeLa-M cells were co-transfected with pAD2TE1 and either an empty vector (pFLAG-CMV-2) or a plasmid that encodes an amino-terminal FLAG-tagged RNase L expression plasmid. Two days after transfection, cells were selected with hygromycin containing medium for an additional 4 days when total cell lysates and L1 RNPs were prepared. Western blotting, using anti-T7 and anti-TAP antibodies, was used to detect ORF1p and ORF2p, respectively. Shown are two exposures of the ORF2p anti-TAP western blot. Endogenous ribosomal S6 protein was used as the loading/transfer control. β-Actin detection discriminated the total cell lysate (left side of panel) from the L1 RNP fractions (right side of panel). The experiments were repeated twice (biological replicates) with similar results. Shown are data from one representative experiment. ( B ) RNase L does not inhibit exogenous EGFP protein expression: HeLa-M cells were co-transfected with pEGFP-C1 and either an empty vector (pFLAG-CMV-2) or a plasmid that encodes an amino-terminal FLAG-tagged RNase L expression plasmid. Total cell lysates were harvested and the expression of RNase L and GFP was detected in western blot experiments using anti-RNase L and anti-GFP antibodies at 48 h after transfection. GAPDH served as a loading and transfer control.

    Journal: Nucleic Acids Research

    Article Title: RNase L restricts the mobility of engineered retrotransposons in cultured human cells

    doi: 10.1093/nar/gkt1308

    Figure Lengend Snippet: Expression of RNase L reduces L1 protein expression. ( A ) L1 protein expression: HeLa-M cells were co-transfected with pAD2TE1 and either an empty vector (pFLAG-CMV-2) or a plasmid that encodes an amino-terminal FLAG-tagged RNase L expression plasmid. Two days after transfection, cells were selected with hygromycin containing medium for an additional 4 days when total cell lysates and L1 RNPs were prepared. Western blotting, using anti-T7 and anti-TAP antibodies, was used to detect ORF1p and ORF2p, respectively. Shown are two exposures of the ORF2p anti-TAP western blot. Endogenous ribosomal S6 protein was used as the loading/transfer control. β-Actin detection discriminated the total cell lysate (left side of panel) from the L1 RNP fractions (right side of panel). The experiments were repeated twice (biological replicates) with similar results. Shown are data from one representative experiment. ( B ) RNase L does not inhibit exogenous EGFP protein expression: HeLa-M cells were co-transfected with pEGFP-C1 and either an empty vector (pFLAG-CMV-2) or a plasmid that encodes an amino-terminal FLAG-tagged RNase L expression plasmid. Total cell lysates were harvested and the expression of RNase L and GFP was detected in western blot experiments using anti-RNase L and anti-GFP antibodies at 48 h after transfection. GAPDH served as a loading and transfer control.

    Article Snippet: For enhanced green fluorescent protein (EGFP)-based retrotransposition assays, HeLa-M cells were transfected with 0.5 µg of an active (pLRE3-mEGFPI ) or inactive (pJM111-LRE3-mEGFPI ) L1 expression plasmid and 0.5 µg of a corresponding RNase L expression plasmid, using 3 µl of Fugene6 transfection reagent (Roche) per well.

    Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot

    Expression of RNase L inhibits L1 retrotransposition in an EGFP -based retrotransposition assay. ( A ) Results from the assays: HeLa-M cells were co-transfected with an expression construct containing an active human L1 (pLRE3- mEGFPI ) and an empty vector (pFLAG-CMV-2), a plasmid encoding an amino-terminal FLAG-tagged RNase L expression plasmid or an amino-terminal HA-tagged A3A expression plasmid. Experiments with a retrotransposition-defective L1 pJM111-LRE3- mEGFPI served as a negative control. The cells were subjected to puromycin selection for 4 days after transfection. Fluorescence Activated Cell Sorting (FACS) was then used to screen for EGFP-positive cells. The X-axis indicates the construct name. The Y-axis indicates the percentage of EGFP-positive cells. For each sample, 2 × 10 5 cells were analyzed and the percentage of EGFP-positive cells was calculated with using the FlowJo software package. Data were analyzed with one-way ANOVA with post hoc tests and are shown as mean ± SD from a single experiment with three technical replicates. * P

    Journal: Nucleic Acids Research

    Article Title: RNase L restricts the mobility of engineered retrotransposons in cultured human cells

    doi: 10.1093/nar/gkt1308

    Figure Lengend Snippet: Expression of RNase L inhibits L1 retrotransposition in an EGFP -based retrotransposition assay. ( A ) Results from the assays: HeLa-M cells were co-transfected with an expression construct containing an active human L1 (pLRE3- mEGFPI ) and an empty vector (pFLAG-CMV-2), a plasmid encoding an amino-terminal FLAG-tagged RNase L expression plasmid or an amino-terminal HA-tagged A3A expression plasmid. Experiments with a retrotransposition-defective L1 pJM111-LRE3- mEGFPI served as a negative control. The cells were subjected to puromycin selection for 4 days after transfection. Fluorescence Activated Cell Sorting (FACS) was then used to screen for EGFP-positive cells. The X-axis indicates the construct name. The Y-axis indicates the percentage of EGFP-positive cells. For each sample, 2 × 10 5 cells were analyzed and the percentage of EGFP-positive cells was calculated with using the FlowJo software package. Data were analyzed with one-way ANOVA with post hoc tests and are shown as mean ± SD from a single experiment with three technical replicates. * P

    Article Snippet: For enhanced green fluorescent protein (EGFP)-based retrotransposition assays, HeLa-M cells were transfected with 0.5 µg of an active (pLRE3-mEGFPI ) or inactive (pJM111-LRE3-mEGFPI ) L1 expression plasmid and 0.5 µg of a corresponding RNase L expression plasmid, using 3 µl of Fugene6 transfection reagent (Roche) per well.

    Techniques: Expressing, Transfection, Construct, Plasmid Preparation, Negative Control, Selection, Fluorescence, FACS, Software

    Inhibition of L1 retrotransposition by RNase L. ( A ) L1 Retrotransposition Assays: HeLa-M cells were co-transfected with pJM101/L1.3 and either an empty vector (pFLAG-CMV-2) or a plasmid that encodes amino-terminal FLAG-tagged versions of the following proteins: RNase L, A3A or RIG-I. The cells were subjected to selection for 10 days and G418-resistant foci were fixed and stained with crystal violet for visualization purposes. A representative tissue culture dish for each condition is shown. ( B ) Quantitation of the L1 Retrotransposition Assays: The X-axis depicts the co-transfected construct names. The Y-axis depicts the number of G418-resistant foci per cell culture dish. Data are shown as the mean ± standard deviation (SD) from a single experiment with three technical replicates. * P

    Journal: Nucleic Acids Research

    Article Title: RNase L restricts the mobility of engineered retrotransposons in cultured human cells

    doi: 10.1093/nar/gkt1308

    Figure Lengend Snippet: Inhibition of L1 retrotransposition by RNase L. ( A ) L1 Retrotransposition Assays: HeLa-M cells were co-transfected with pJM101/L1.3 and either an empty vector (pFLAG-CMV-2) or a plasmid that encodes amino-terminal FLAG-tagged versions of the following proteins: RNase L, A3A or RIG-I. The cells were subjected to selection for 10 days and G418-resistant foci were fixed and stained with crystal violet for visualization purposes. A representative tissue culture dish for each condition is shown. ( B ) Quantitation of the L1 Retrotransposition Assays: The X-axis depicts the co-transfected construct names. The Y-axis depicts the number of G418-resistant foci per cell culture dish. Data are shown as the mean ± standard deviation (SD) from a single experiment with three technical replicates. * P

    Article Snippet: For enhanced green fluorescent protein (EGFP)-based retrotransposition assays, HeLa-M cells were transfected with 0.5 µg of an active (pLRE3-mEGFPI ) or inactive (pJM111-LRE3-mEGFPI ) L1 expression plasmid and 0.5 µg of a corresponding RNase L expression plasmid, using 3 µl of Fugene6 transfection reagent (Roche) per well.

    Techniques: Inhibition, Transfection, Plasmid Preparation, Selection, Staining, Quantitation Assay, Construct, Cell Culture, Standard Deviation

    An overview of the L1 and IAP retrotransposition assays. ( A ) Schematics of L1 and IAP constructs: The L1 and IAP constructs contain a NEO-based ( mneoI ) or EGFP -based ( mEGFPI ) retrotransposition indicator cassette near their 3′ ends. The indicator cassettes are in an anti-sense (backward) orientation relative to the transcriptional orientation of the L1 or IAP elements. The indicator cassettes also contain an intron that is in the same transcriptional orientation as the retroelement. SD and SA indicate the splice donor and splice acceptor sites of the intron, respectively. Pr′ indicates the promoter driving the expression of the retrotransposition indicator cassette. Closed lollipops indicate the polyadenylation signal on the indicator cassette. A CMV promoter enhances the expression of the pJM101/L1.3, pAD2TE1, pES2TE1 and pAD3TE1 L1 vectors. An SV40 polyadenylation signal is present at the 3′ end of each L1 expression cassette. Notably, the mneoI -based L1 vectors are expressed from a pCEP4 vector that contains a HYG and an EBNA-1 gene. The mEGFPI-based L1 vectors are expressed from a pCEP4 vector that was modified to contain a PURO gene; it also contains the EBNA-1 gene. Flag symbols indicate the names of epitope-tags present in some L1 vectors. The SP and ASP labels indicate the sense and anti-sense promoters located in the L1 5′-UTR. The MS2 24x designation indicates the 24 copies of the MS2-GFP RNA binding motif in the pAD3TE1 construct. The PCR primers for pAD2TE1 are labeled F1, R1, F2 and R2 (see ‘Materials and Methods’ section for details). In the IAP vector [pDJ33/440N1 neo TNF ( 13 )], Pr indicates the viral LTR promoter. The IAP GAG and POL genes also are indicated. ( B ) Rationale of the assay: Transcription from a promoter driving L1 or IAP expression allows splicing of the intron from either the mneoI- or EGFP-based indicator cassettes. Retrotransposition of the resultant RNA leads to activation of the reporter gene, conferring either G418-resistance or EGFP-positivity to host cells. TSD indicates a target site duplication flanking the retrotransposed L1. ( C ) Experimental protocols to detect L1 retrotransposition: Cells were co-transfected with an engineered L1 or IAP retroelement and either an empty vector (pFLAG-CMV-2) or amino-terminal FLAG-tagged RNase L expression plasmid. For the mneoI -based assays, the transfected cells were subjected to G418 selection 2 days after transfection. The numbers of G418-resistant foci serve as a readout of retrotransposition efficiency. For the mEGFPI -based assays, FACS analysis was used to measure the percentage of EGFP-positive cells 4 days after transfection (See ‘Materials and Methods’ section for further details about each assay).

    Journal: Nucleic Acids Research

    Article Title: RNase L restricts the mobility of engineered retrotransposons in cultured human cells

    doi: 10.1093/nar/gkt1308

    Figure Lengend Snippet: An overview of the L1 and IAP retrotransposition assays. ( A ) Schematics of L1 and IAP constructs: The L1 and IAP constructs contain a NEO-based ( mneoI ) or EGFP -based ( mEGFPI ) retrotransposition indicator cassette near their 3′ ends. The indicator cassettes are in an anti-sense (backward) orientation relative to the transcriptional orientation of the L1 or IAP elements. The indicator cassettes also contain an intron that is in the same transcriptional orientation as the retroelement. SD and SA indicate the splice donor and splice acceptor sites of the intron, respectively. Pr′ indicates the promoter driving the expression of the retrotransposition indicator cassette. Closed lollipops indicate the polyadenylation signal on the indicator cassette. A CMV promoter enhances the expression of the pJM101/L1.3, pAD2TE1, pES2TE1 and pAD3TE1 L1 vectors. An SV40 polyadenylation signal is present at the 3′ end of each L1 expression cassette. Notably, the mneoI -based L1 vectors are expressed from a pCEP4 vector that contains a HYG and an EBNA-1 gene. The mEGFPI-based L1 vectors are expressed from a pCEP4 vector that was modified to contain a PURO gene; it also contains the EBNA-1 gene. Flag symbols indicate the names of epitope-tags present in some L1 vectors. The SP and ASP labels indicate the sense and anti-sense promoters located in the L1 5′-UTR. The MS2 24x designation indicates the 24 copies of the MS2-GFP RNA binding motif in the pAD3TE1 construct. The PCR primers for pAD2TE1 are labeled F1, R1, F2 and R2 (see ‘Materials and Methods’ section for details). In the IAP vector [pDJ33/440N1 neo TNF ( 13 )], Pr indicates the viral LTR promoter. The IAP GAG and POL genes also are indicated. ( B ) Rationale of the assay: Transcription from a promoter driving L1 or IAP expression allows splicing of the intron from either the mneoI- or EGFP-based indicator cassettes. Retrotransposition of the resultant RNA leads to activation of the reporter gene, conferring either G418-resistance or EGFP-positivity to host cells. TSD indicates a target site duplication flanking the retrotransposed L1. ( C ) Experimental protocols to detect L1 retrotransposition: Cells were co-transfected with an engineered L1 or IAP retroelement and either an empty vector (pFLAG-CMV-2) or amino-terminal FLAG-tagged RNase L expression plasmid. For the mneoI -based assays, the transfected cells were subjected to G418 selection 2 days after transfection. The numbers of G418-resistant foci serve as a readout of retrotransposition efficiency. For the mEGFPI -based assays, FACS analysis was used to measure the percentage of EGFP-positive cells 4 days after transfection (See ‘Materials and Methods’ section for further details about each assay).

    Article Snippet: For enhanced green fluorescent protein (EGFP)-based retrotransposition assays, HeLa-M cells were transfected with 0.5 µg of an active (pLRE3-mEGFPI ) or inactive (pJM111-LRE3-mEGFPI ) L1 expression plasmid and 0.5 µg of a corresponding RNase L expression plasmid, using 3 µl of Fugene6 transfection reagent (Roche) per well.

    Techniques: Construct, Expressing, Plasmid Preparation, Modification, RNA Binding Assay, Polymerase Chain Reaction, Labeling, Activation Assay, Transfection, Selection, FACS

    RNase L reduces L1 RNA accumulation in cells. ( A ) Results of qRT-PCR experiments: HeLa-M cells were co-transfected with pAD2TE1 and an empty vector (pFLAG-CMV-2) or an amino-terminal FLAG-tagged RNase L expression plasmid. L1 RNA levels were determined 48 h after transfection using the Sybr Green method ( 84 ). The X-axis indicates the RNase L co-transfected samples. The Y-axis indicates the relative expression level of L1 RNA from the transfected construct. The L1 RNA amounts were normalized with hygromycin mRNA levels (see ‘Materials and Methods’ section for detailed PCR strategy). Data are represented as the mean ± SD from three technical replicates of a single representative experiment. * P

    Journal: Nucleic Acids Research

    Article Title: RNase L restricts the mobility of engineered retrotransposons in cultured human cells

    doi: 10.1093/nar/gkt1308

    Figure Lengend Snippet: RNase L reduces L1 RNA accumulation in cells. ( A ) Results of qRT-PCR experiments: HeLa-M cells were co-transfected with pAD2TE1 and an empty vector (pFLAG-CMV-2) or an amino-terminal FLAG-tagged RNase L expression plasmid. L1 RNA levels were determined 48 h after transfection using the Sybr Green method ( 84 ). The X-axis indicates the RNase L co-transfected samples. The Y-axis indicates the relative expression level of L1 RNA from the transfected construct. The L1 RNA amounts were normalized with hygromycin mRNA levels (see ‘Materials and Methods’ section for detailed PCR strategy). Data are represented as the mean ± SD from three technical replicates of a single representative experiment. * P

    Article Snippet: For enhanced green fluorescent protein (EGFP)-based retrotransposition assays, HeLa-M cells were transfected with 0.5 µg of an active (pLRE3-mEGFPI ) or inactive (pJM111-LRE3-mEGFPI ) L1 expression plasmid and 0.5 µg of a corresponding RNase L expression plasmid, using 3 µl of Fugene6 transfection reagent (Roche) per well.

    Techniques: Quantitative RT-PCR, Transfection, Plasmid Preparation, Expressing, SYBR Green Assay, Construct, Polymerase Chain Reaction

    Inhibition of IAP retrotransposition by RNase L. ( A ) IAP Retrotransposition Assays: HeLa-M cells were co-transfected with a mouse IAP expression construct (pDJ33/440N1 neo TNF ) and either an empty vector (pFLAG-CMV-2) or an expression plasmid that encodes an amino-terminal FLAG-tagged version of the following proteins: WT RNase L, a catalytically inactive RNase L mutant (R667A), a constitutively active RNase L mutant (NΔ385), A3A or RIG-I. The cells were subject to selection for 10 days and G418-resistant foci were fixed and stained with crystal violet for visualization purposes. A representative tissue culture dish for each condition is shown. ( B ) Quantitation of the IAP Retrotransposition Assays: The X-axis depicts names of constructs co-transfected into cells with the IAP construct. The Y-axis depicts the number of G418-resistant foci per cell culture dish. Data are represented as the mean ± standard deviation (SD) from a single experiment with three technical replicates. * P

    Journal: Nucleic Acids Research

    Article Title: RNase L restricts the mobility of engineered retrotransposons in cultured human cells

    doi: 10.1093/nar/gkt1308

    Figure Lengend Snippet: Inhibition of IAP retrotransposition by RNase L. ( A ) IAP Retrotransposition Assays: HeLa-M cells were co-transfected with a mouse IAP expression construct (pDJ33/440N1 neo TNF ) and either an empty vector (pFLAG-CMV-2) or an expression plasmid that encodes an amino-terminal FLAG-tagged version of the following proteins: WT RNase L, a catalytically inactive RNase L mutant (R667A), a constitutively active RNase L mutant (NΔ385), A3A or RIG-I. The cells were subject to selection for 10 days and G418-resistant foci were fixed and stained with crystal violet for visualization purposes. A representative tissue culture dish for each condition is shown. ( B ) Quantitation of the IAP Retrotransposition Assays: The X-axis depicts names of constructs co-transfected into cells with the IAP construct. The Y-axis depicts the number of G418-resistant foci per cell culture dish. Data are represented as the mean ± standard deviation (SD) from a single experiment with three technical replicates. * P

    Article Snippet: For enhanced green fluorescent protein (EGFP)-based retrotransposition assays, HeLa-M cells were transfected with 0.5 µg of an active (pLRE3-mEGFPI ) or inactive (pJM111-LRE3-mEGFPI ) L1 expression plasmid and 0.5 µg of a corresponding RNase L expression plasmid, using 3 µl of Fugene6 transfection reagent (Roche) per well.

    Techniques: Inhibition, Transfection, Expressing, Construct, Plasmid Preparation, Mutagenesis, Selection, Staining, Quantitation Assay, Cell Culture, Standard Deviation

    Depletion of endogenous RNase L increases L1 retrotransposition efficiency. ( A ) Knockdown of endogenous RNase L protein: Hey1b cells were transfected with control siRNA pools or RNase L siRNA pools. Western blotting using an anti-RNase L monoclonal antibody confirmed RNase L knockdown 48 h after siRNA transfection. β-Actin served as loading and transfer control. The band intensity was quantified with ImageJ software ( 83 ) and the relative ratio of RNase L to β-actin is shown. ( B ) Representative Retrotransposition Assay Results: Control siRNA (control) and siRNA-mediated RNase L depleted cells (RNase L) were transfected with either pLRE3- mEGFPI or pJM111-LRE3- mEGFPI . L1 retrotransposition was assayed as described in Figure 4 . Representative FACS plots are shown, as is the conservative gating strategy used to detect EGFP-positive cells. ( C ) Quantitation of the Retrotransposition Assays: The X-axis indicates the control siRNA (control) or siRNA-mediated RNase L depleted cells (RNase L). The Y-axis indicates the percentage of EGFP-positive cells. For each sample, 2 × 10 5 cells were analyzed and the percentage of EGFP-positive cells was calculated with using the FlowJo software package. The experiment was conducted four times (biological replicates) with similar results; representative data from one experiment are shown. Data are reported as the mean ± SD from three technical replicates of a single representative experiment. The asterisk indicates a P = 0.0079 and was calculated with two-tailed Student’s t -test.

    Journal: Nucleic Acids Research

    Article Title: RNase L restricts the mobility of engineered retrotransposons in cultured human cells

    doi: 10.1093/nar/gkt1308

    Figure Lengend Snippet: Depletion of endogenous RNase L increases L1 retrotransposition efficiency. ( A ) Knockdown of endogenous RNase L protein: Hey1b cells were transfected with control siRNA pools or RNase L siRNA pools. Western blotting using an anti-RNase L monoclonal antibody confirmed RNase L knockdown 48 h after siRNA transfection. β-Actin served as loading and transfer control. The band intensity was quantified with ImageJ software ( 83 ) and the relative ratio of RNase L to β-actin is shown. ( B ) Representative Retrotransposition Assay Results: Control siRNA (control) and siRNA-mediated RNase L depleted cells (RNase L) were transfected with either pLRE3- mEGFPI or pJM111-LRE3- mEGFPI . L1 retrotransposition was assayed as described in Figure 4 . Representative FACS plots are shown, as is the conservative gating strategy used to detect EGFP-positive cells. ( C ) Quantitation of the Retrotransposition Assays: The X-axis indicates the control siRNA (control) or siRNA-mediated RNase L depleted cells (RNase L). The Y-axis indicates the percentage of EGFP-positive cells. For each sample, 2 × 10 5 cells were analyzed and the percentage of EGFP-positive cells was calculated with using the FlowJo software package. The experiment was conducted four times (biological replicates) with similar results; representative data from one experiment are shown. Data are reported as the mean ± SD from three technical replicates of a single representative experiment. The asterisk indicates a P = 0.0079 and was calculated with two-tailed Student’s t -test.

    Article Snippet: For enhanced green fluorescent protein (EGFP)-based retrotransposition assays, HeLa-M cells were transfected with 0.5 µg of an active (pLRE3-mEGFPI ) or inactive (pJM111-LRE3-mEGFPI ) L1 expression plasmid and 0.5 µg of a corresponding RNase L expression plasmid, using 3 µl of Fugene6 transfection reagent (Roche) per well.

    Techniques: Transfection, Western Blot, Software, FACS, Quantitation Assay, Two Tailed Test