rnase h  (New England Biolabs)


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    RNase H
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    RNase H 1 250 units
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    New England Biolabs rnase h
    RNase H
    RNase H 1 250 units
    https://www.bioz.com/result/rnase h/product/New England Biolabs
    Average 99 stars, based on 112 article reviews
    Price from $9.99 to $1999.99
    rnase h - by Bioz Stars, 2020-10
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    Images

    1) Product Images from "Translation initiation of alphavirus mRNA reveals new insights into the topology of the 48S initiation complex"

    Article Title: Translation initiation of alphavirus mRNA reveals new insights into the topology of the 48S initiation complex

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky071

    eIF4A activity within the 48S complex. ( A ) RNase H-mapping of RNA–RNA interactions between SV-DLP U1 and 18S rRNA. The analysis was carried out in the absence or presence of 1 μM hippuristanol, with identification of the resulting RNA fragments indicated. For clarity, a schematic diagram of the ES6S and h16–18 regions of rabbit 18S rRNA with the primers used for RNase H digestion is shown. The use of oligos 4 and 9 limited the region of 18S rRNA (509–830) where the crosslinkings concentrated. Bands corresponding to crosslinking of SV DLP U1 mRNA with the ES6S region (680–1863) and h16-h18 helices (1–662) were quantified by densitometry and expressed as a ratio. Data are the mean ± SEM from four independent experiments. ( B ). Reactivity to SHAPE reagent (NMIA) is higher for unpaired nucleotides (red) and low for those involved in pairings (black). Stops corresponding to toeprints are marked with arrowheads. Quantification of toeprint ratios (17–19/23–25) in absence or presence of hippuristanol is shown from three independent experiments; data are the mean ± SEM.
    Figure Legend Snippet: eIF4A activity within the 48S complex. ( A ) RNase H-mapping of RNA–RNA interactions between SV-DLP U1 and 18S rRNA. The analysis was carried out in the absence or presence of 1 μM hippuristanol, with identification of the resulting RNA fragments indicated. For clarity, a schematic diagram of the ES6S and h16–18 regions of rabbit 18S rRNA with the primers used for RNase H digestion is shown. The use of oligos 4 and 9 limited the region of 18S rRNA (509–830) where the crosslinkings concentrated. Bands corresponding to crosslinking of SV DLP U1 mRNA with the ES6S region (680–1863) and h16-h18 helices (1–662) were quantified by densitometry and expressed as a ratio. Data are the mean ± SEM from four independent experiments. ( B ). Reactivity to SHAPE reagent (NMIA) is higher for unpaired nucleotides (red) and low for those involved in pairings (black). Stops corresponding to toeprints are marked with arrowheads. Quantification of toeprint ratios (17–19/23–25) in absence or presence of hippuristanol is shown from three independent experiments; data are the mean ± SEM.

    Techniques Used: Activity Assay

    2) Product Images from "A Eukaryotic Translation Initiation Factor 4E-Binding Protein Promotes mRNA Decapping and Is Required for PUF Repression"

    Article Title: A Eukaryotic Translation Initiation Factor 4E-Binding Protein Promotes mRNA Decapping and Is Required for PUF Repression

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00483-12

    Eap1p promotes decapping of HO mRNA. (A) HO mRNA was cleaved with RNase H and a DNA oligonucleotide to produce a 1,600-nucleotide 5′ fragment and a 253-nucleotide 3′ fragment with a poly(A) tail of up to 80 adenosines (pA 80 ). (B) Northern
    Figure Legend Snippet: Eap1p promotes decapping of HO mRNA. (A) HO mRNA was cleaved with RNase H and a DNA oligonucleotide to produce a 1,600-nucleotide 5′ fragment and a 253-nucleotide 3′ fragment with a poly(A) tail of up to 80 adenosines (pA 80 ). (B) Northern

    Techniques Used: Northern Blot

    3) Product Images from "Defining the location of promoter-associated R-loops at near-nucleotide resolution using bisDRIP-seq"

    Article Title: Defining the location of promoter-associated R-loops at near-nucleotide resolution using bisDRIP-seq

    Journal: eLife

    doi: 10.7554/eLife.28306

    bisDRIP-seq scores are sensitive to RNase H. ( A ) RNA-DNA hybrid formation is necessary for enrichment of bisDRIP-seq signal at sites of R-loop formation. RNA-DNA hybrids are integral components of R-loops. RNA-DNA hybrids are known to bind the S9.6 antibody and to be degraded by RNase H. R-loop signal should therefore be lost in the absence of S9.6 immunoprecipitation or if the sample is treated with RNase H. In each of these plots, the template strand refers to the strand used for RPPH1 transcription, rather than the template strand used for PAPR2 transcription. Also, in each plot the bisDRIP-seq score on the template strand and non-template strand were plotted below the x-axis (orange) or above the x-axis (blue), respectively. In the top plot, bisDRIP-seq scores were mapped to the genomic region surrounding RPPH1 from ‘input’ samples (mean bisDRIP-seq score from n = 2 samples). These input samples received the entire bisDRIP-seq treatment except for the S9.6 immunoprecipitation enrichment steps. In the middle plot, bisDRIP-seq scores were mapped to the genomic region surrounding RPPH1 from a control-treated bisDRIP-seq experiment that was not treated with RNase H (n = 1 sample). In the bottom plot, bisDRIP-seq scores were mapped to the genomic region surrounding RPPH1 from an RNase H-treated bisDRIP-seq experiment (n = 1 sample). The bisDRIP-seq sample that was not treated with RNase H has high bisDRIP-seq score at the RPPH1 locus. On the other hand, there is a large decrease in bisDRIP-seq signal in the sample treated with RNase H. This suggests that bisDRIP-seq signal in the RPPH1 locus depends on the presence of an RNA-DNA hybrid.
    Figure Legend Snippet: bisDRIP-seq scores are sensitive to RNase H. ( A ) RNA-DNA hybrid formation is necessary for enrichment of bisDRIP-seq signal at sites of R-loop formation. RNA-DNA hybrids are integral components of R-loops. RNA-DNA hybrids are known to bind the S9.6 antibody and to be degraded by RNase H. R-loop signal should therefore be lost in the absence of S9.6 immunoprecipitation or if the sample is treated with RNase H. In each of these plots, the template strand refers to the strand used for RPPH1 transcription, rather than the template strand used for PAPR2 transcription. Also, in each plot the bisDRIP-seq score on the template strand and non-template strand were plotted below the x-axis (orange) or above the x-axis (blue), respectively. In the top plot, bisDRIP-seq scores were mapped to the genomic region surrounding RPPH1 from ‘input’ samples (mean bisDRIP-seq score from n = 2 samples). These input samples received the entire bisDRIP-seq treatment except for the S9.6 immunoprecipitation enrichment steps. In the middle plot, bisDRIP-seq scores were mapped to the genomic region surrounding RPPH1 from a control-treated bisDRIP-seq experiment that was not treated with RNase H (n = 1 sample). In the bottom plot, bisDRIP-seq scores were mapped to the genomic region surrounding RPPH1 from an RNase H-treated bisDRIP-seq experiment (n = 1 sample). The bisDRIP-seq sample that was not treated with RNase H has high bisDRIP-seq score at the RPPH1 locus. On the other hand, there is a large decrease in bisDRIP-seq signal in the sample treated with RNase H. This suggests that bisDRIP-seq signal in the RPPH1 locus depends on the presence of an RNA-DNA hybrid.

    Techniques Used: Immunoprecipitation

    The strand asymmetry of bisDRIP-seq scores 3' of the transcription start site is sensitive to RNase H. ( A,B ) The strand asymmetry in bisDRIP-seq scores 3' of transcription start sites is not observed after RNase H treatment. RNA-DNA hybrids are core components of R-loops. If the strand asymmetry observed in metaplots of promoter regions is due to R-loops, than it should be selectively sensitive to RNase H. We therefore generated metaplots of bisDRIP-seq scores from two samples treated identically except that ( A ) received a vehicle treatment, while ( B ) was treated with RNase H. Metaplots were created by summing the bisDRIP-seq scores across active promoter regions (n = 15644) at each nucleotide position relative to the transcription start site. bisDRIP-seq scores were calculated separately for the nucleotide position on the non-template strand (blue) and template strand (orange). In the vehicle-treated sample, non-template bisDRIP-seq scores are higher than template bisDRIP-seq scores immediately 3' of the transcription start site. This replicates the result from Figure 3E . On the other hand, there is a complete loss of bisDRIP-seq score strand asymmetry after RNase H treatment in ( B ). This confirms that the asymmetry observed in Figure 3E is caused by R-loops.
    Figure Legend Snippet: The strand asymmetry of bisDRIP-seq scores 3' of the transcription start site is sensitive to RNase H. ( A,B ) The strand asymmetry in bisDRIP-seq scores 3' of transcription start sites is not observed after RNase H treatment. RNA-DNA hybrids are core components of R-loops. If the strand asymmetry observed in metaplots of promoter regions is due to R-loops, than it should be selectively sensitive to RNase H. We therefore generated metaplots of bisDRIP-seq scores from two samples treated identically except that ( A ) received a vehicle treatment, while ( B ) was treated with RNase H. Metaplots were created by summing the bisDRIP-seq scores across active promoter regions (n = 15644) at each nucleotide position relative to the transcription start site. bisDRIP-seq scores were calculated separately for the nucleotide position on the non-template strand (blue) and template strand (orange). In the vehicle-treated sample, non-template bisDRIP-seq scores are higher than template bisDRIP-seq scores immediately 3' of the transcription start site. This replicates the result from Figure 3E . On the other hand, there is a complete loss of bisDRIP-seq score strand asymmetry after RNase H treatment in ( B ). This confirms that the asymmetry observed in Figure 3E is caused by R-loops.

    Techniques Used: Generated

    R-loop signal in MALAT1 and NEAT1 is reduced by RNase H treatment. ( A,B ) bisDRIP-seq enrichment at ( A ) MALAT1 and ( B ) NEAT1 is lost after RNase H treatment and is not observed without RNA-DNA hybrid immunoprecipitation by S9.6 antibody. RNase H should selectively degrade the RNA component of R-loops. Thus, if bisDRIP-seq signal is caused by R-loops, RNase H treatment should reduce bisDRIP-seq signal. In the top panel, bisDRIP-seq scores from ‘input’ samples (mean of n = 2 samples) were mapped to each gene locus. bisDRIP-seq input samples were treated an identical manner to other bisDRIP-seq samples, except that no S9.6 antibody immunoprecipitation steps were performed. In the middle and lower plots, the bisDRIP-seq scores from two matched bisDRIP-seq experiments were mapped to each locus. The only difference between these experiments is that one sample was not treated with RNase H (middle plots, n = 1 sample), while the other sample was treated with RNase H (lower plots, n = 1 sample). At both loci, the sample that was not treated with RNase H replicated the bisDRIP-seq signal observed in standard bisDRIP-seq control experiments ( Figure 6A and B ). On the other hand, there is less bisDRIP-seq signal observed at each loci in both the input and RNase H-treated samples. These results suggest that RNA-DNA hybrids form in the MALAT1 and NEAT1 loci and that these RNA-DNA hybrids cause the high bisDRIP-seq scores observed at these loci. The location of the transcription start site is demarcated by ‘TSS’ and a dashed vertical line.
    Figure Legend Snippet: R-loop signal in MALAT1 and NEAT1 is reduced by RNase H treatment. ( A,B ) bisDRIP-seq enrichment at ( A ) MALAT1 and ( B ) NEAT1 is lost after RNase H treatment and is not observed without RNA-DNA hybrid immunoprecipitation by S9.6 antibody. RNase H should selectively degrade the RNA component of R-loops. Thus, if bisDRIP-seq signal is caused by R-loops, RNase H treatment should reduce bisDRIP-seq signal. In the top panel, bisDRIP-seq scores from ‘input’ samples (mean of n = 2 samples) were mapped to each gene locus. bisDRIP-seq input samples were treated an identical manner to other bisDRIP-seq samples, except that no S9.6 antibody immunoprecipitation steps were performed. In the middle and lower plots, the bisDRIP-seq scores from two matched bisDRIP-seq experiments were mapped to each locus. The only difference between these experiments is that one sample was not treated with RNase H (middle plots, n = 1 sample), while the other sample was treated with RNase H (lower plots, n = 1 sample). At both loci, the sample that was not treated with RNase H replicated the bisDRIP-seq signal observed in standard bisDRIP-seq control experiments ( Figure 6A and B ). On the other hand, there is less bisDRIP-seq signal observed at each loci in both the input and RNase H-treated samples. These results suggest that RNA-DNA hybrids form in the MALAT1 and NEAT1 loci and that these RNA-DNA hybrids cause the high bisDRIP-seq scores observed at these loci. The location of the transcription start site is demarcated by ‘TSS’ and a dashed vertical line.

    Techniques Used: Immunoprecipitation

    Histone genes are enriched in R-loops. ( A,B ) RNA polymerase II binding and RNA levels in the nucleus do not appear to explain the association between histone genes and R-loop formation. R-loop formation was calculated for each promoter region by taking the sum of bisDRIP-seq score strand asymmetry and the triptolide-sensitivity the non-template bisDRIP-seq scores. R-loop formation was then plotted against ( A ) nuclear RNA levels or ( B ) POLR2A (RNA polymerase II) chromatin immunoprecipitation (ChIP) signal for each promoter region. Promoter regions were plotted separately for non-histone genes (blue) and replication-dependent histone genes (orange). A linear regression for all genes was also plotted (blue). Both nuclear RNA levels and RNA polymerase II ChIP signal were weakly correlated with R-loop formation. Nevertheless, replication-dependent histone genes typically had higher R-loop formation scores than genes with similar promoter activity. This suggests that the nuclear RNA level of histones mRNAs in do not explain the high R-loop signal of these genes. It also suggests that the level of RNA polymerase II bound to histone genes does not explain the high R-loop signal observed at these genes. The bisDRIP-seq values in both plots were derived from the mean bisDRIP-seq scores of n = 13 control-treated samples and mean bisDRIP-seq scores of n = 2 triptolide-treated samples. In ( A ), we examined n = 1813 non-histone genes and n = 2 histone genes. In ( B ), we examined n = 2064 non-histone genes and n = 13 histone genes. TPKM refers to transcripts per kilobase million. The shaded areas around linear regression models represent 95% confidence intervals. ( C ) RNase H sensitivity of the bisDRIP-seq signal in histone genes. Metaplots were generated of histone gene non-template bisDRIP-seq score from three different bisDRIP-seq experiments. In the top plot, bisDRIP-seq scores were derived from ‘input’ samples (mean bisDRIP-seq score from n = 2 samples). These input samples received the entire bisDRIP-seq treatment except for the S9.6 immunoprecipitation enrichment steps. In the middle plot, bisDRIP-seq scores were derived from a bisDRIP-seq experiment that did not include an RNase H treatment (n = 1 sample). In the bottom plot, bisDRIP-seq scores were derived from an RNase H-treated bisDRIP-seq experiment (n = 1 sample). The bisDRIP-seq sample that was not treated with RNase H replicated the high non-template bisDRIP-seq scores observed in other bisDRIP-seq experiments at histone loci. On the other hand, almost no bisDRIP-seq signal was observed in either the input samples or in the bisDRIP-seq sample treated with RNase H. This suggests that the bisDRIP-seq signal observed at these gene loci is caused by R-loops. Transcription start sites are indicated by both ‘TSS’ and by dashed vertical lines. ( D,E ) bisDRIP-seq enrichment at ( D ) HIST1H2BG and ( E ) HIST1H1E is due to the presence of R-loops. In the middle and top plots, we compare the bisDRIP-seq from an RNase H treated sample (n = 1) and a bisDRIP-seq sample that was not treated with RNase H (n = 1), respectively. In both the middle and top plot, bisDRIP-seq scores from each experiment were mapped to the genomic region surrounding ( D ) HIST1H2BG and ( E ) HIST1H1E . At both loci, the bisDRIP-seq sample that was not treated with RNase H had similar bisDRIP-seq signal to our other bisDRIP-seq control experiments ( Figure 5D and E ). On the other hand, there is a clear reduction in bisDRIP-seq scores in the RNase H-treated sample. This decrease in signal after RNase H treatment suggests that RNA-DNA hybrids do form at these loci. In the bottom panel, the percentage of each nucleotide is plotted in 51 bp windows. There is no obvious relationship between nucleotide composition and bisDRIP-seq scores throughout these two genes.
    Figure Legend Snippet: Histone genes are enriched in R-loops. ( A,B ) RNA polymerase II binding and RNA levels in the nucleus do not appear to explain the association between histone genes and R-loop formation. R-loop formation was calculated for each promoter region by taking the sum of bisDRIP-seq score strand asymmetry and the triptolide-sensitivity the non-template bisDRIP-seq scores. R-loop formation was then plotted against ( A ) nuclear RNA levels or ( B ) POLR2A (RNA polymerase II) chromatin immunoprecipitation (ChIP) signal for each promoter region. Promoter regions were plotted separately for non-histone genes (blue) and replication-dependent histone genes (orange). A linear regression for all genes was also plotted (blue). Both nuclear RNA levels and RNA polymerase II ChIP signal were weakly correlated with R-loop formation. Nevertheless, replication-dependent histone genes typically had higher R-loop formation scores than genes with similar promoter activity. This suggests that the nuclear RNA level of histones mRNAs in do not explain the high R-loop signal of these genes. It also suggests that the level of RNA polymerase II bound to histone genes does not explain the high R-loop signal observed at these genes. The bisDRIP-seq values in both plots were derived from the mean bisDRIP-seq scores of n = 13 control-treated samples and mean bisDRIP-seq scores of n = 2 triptolide-treated samples. In ( A ), we examined n = 1813 non-histone genes and n = 2 histone genes. In ( B ), we examined n = 2064 non-histone genes and n = 13 histone genes. TPKM refers to transcripts per kilobase million. The shaded areas around linear regression models represent 95% confidence intervals. ( C ) RNase H sensitivity of the bisDRIP-seq signal in histone genes. Metaplots were generated of histone gene non-template bisDRIP-seq score from three different bisDRIP-seq experiments. In the top plot, bisDRIP-seq scores were derived from ‘input’ samples (mean bisDRIP-seq score from n = 2 samples). These input samples received the entire bisDRIP-seq treatment except for the S9.6 immunoprecipitation enrichment steps. In the middle plot, bisDRIP-seq scores were derived from a bisDRIP-seq experiment that did not include an RNase H treatment (n = 1 sample). In the bottom plot, bisDRIP-seq scores were derived from an RNase H-treated bisDRIP-seq experiment (n = 1 sample). The bisDRIP-seq sample that was not treated with RNase H replicated the high non-template bisDRIP-seq scores observed in other bisDRIP-seq experiments at histone loci. On the other hand, almost no bisDRIP-seq signal was observed in either the input samples or in the bisDRIP-seq sample treated with RNase H. This suggests that the bisDRIP-seq signal observed at these gene loci is caused by R-loops. Transcription start sites are indicated by both ‘TSS’ and by dashed vertical lines. ( D,E ) bisDRIP-seq enrichment at ( D ) HIST1H2BG and ( E ) HIST1H1E is due to the presence of R-loops. In the middle and top plots, we compare the bisDRIP-seq from an RNase H treated sample (n = 1) and a bisDRIP-seq sample that was not treated with RNase H (n = 1), respectively. In both the middle and top plot, bisDRIP-seq scores from each experiment were mapped to the genomic region surrounding ( D ) HIST1H2BG and ( E ) HIST1H1E . At both loci, the bisDRIP-seq sample that was not treated with RNase H had similar bisDRIP-seq signal to our other bisDRIP-seq control experiments ( Figure 5D and E ). On the other hand, there is a clear reduction in bisDRIP-seq scores in the RNase H-treated sample. This decrease in signal after RNase H treatment suggests that RNA-DNA hybrids do form at these loci. In the bottom panel, the percentage of each nucleotide is plotted in 51 bp windows. There is no obvious relationship between nucleotide composition and bisDRIP-seq scores throughout these two genes.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Activity Assay, Derivative Assay, Generated, Immunoprecipitation

    4) Product Images from "Defining the location of promoter-associated R-loops at near-nucleotide resolution using bisDRIP-seq"

    Article Title: Defining the location of promoter-associated R-loops at near-nucleotide resolution using bisDRIP-seq

    Journal: eLife

    doi: 10.7554/eLife.28306

    bisDRIP-seq scores are sensitive to RNase H. ( A ) RNA-DNA hybrid formation is necessary for enrichment of bisDRIP-seq signal at sites of R-loop formation. RNA-DNA hybrids are integral components of R-loops. RNA-DNA hybrids are known to bind the S9.6 antibody and to be degraded by RNase H. R-loop signal should therefore be lost in the absence of S9.6 immunoprecipitation or if the sample is treated with RNase H. In each of these plots, the template strand refers to the strand used for RPPH1 transcription, rather than the template strand used for PAPR2 transcription. Also, in each plot the bisDRIP-seq score on the template strand and non-template strand were plotted below the x-axis (orange) or above the x-axis (blue), respectively. In the top plot, bisDRIP-seq scores were mapped to the genomic region surrounding RPPH1 from ‘input’ samples (mean bisDRIP-seq score from n = 2 samples). These input samples received the entire bisDRIP-seq treatment except for the S9.6 immunoprecipitation enrichment steps. In the middle plot, bisDRIP-seq scores were mapped to the genomic region surrounding RPPH1 from a control-treated bisDRIP-seq experiment that was not treated with RNase H (n = 1 sample). In the bottom plot, bisDRIP-seq scores were mapped to the genomic region surrounding RPPH1 from an RNase H-treated bisDRIP-seq experiment (n = 1 sample). The bisDRIP-seq sample that was not treated with RNase H has high bisDRIP-seq score at the RPPH1 locus. On the other hand, there is a large decrease in bisDRIP-seq signal in the sample treated with RNase H. This suggests that bisDRIP-seq signal in the RPPH1 locus depends on the presence of an RNA-DNA hybrid.
    Figure Legend Snippet: bisDRIP-seq scores are sensitive to RNase H. ( A ) RNA-DNA hybrid formation is necessary for enrichment of bisDRIP-seq signal at sites of R-loop formation. RNA-DNA hybrids are integral components of R-loops. RNA-DNA hybrids are known to bind the S9.6 antibody and to be degraded by RNase H. R-loop signal should therefore be lost in the absence of S9.6 immunoprecipitation or if the sample is treated with RNase H. In each of these plots, the template strand refers to the strand used for RPPH1 transcription, rather than the template strand used for PAPR2 transcription. Also, in each plot the bisDRIP-seq score on the template strand and non-template strand were plotted below the x-axis (orange) or above the x-axis (blue), respectively. In the top plot, bisDRIP-seq scores were mapped to the genomic region surrounding RPPH1 from ‘input’ samples (mean bisDRIP-seq score from n = 2 samples). These input samples received the entire bisDRIP-seq treatment except for the S9.6 immunoprecipitation enrichment steps. In the middle plot, bisDRIP-seq scores were mapped to the genomic region surrounding RPPH1 from a control-treated bisDRIP-seq experiment that was not treated with RNase H (n = 1 sample). In the bottom plot, bisDRIP-seq scores were mapped to the genomic region surrounding RPPH1 from an RNase H-treated bisDRIP-seq experiment (n = 1 sample). The bisDRIP-seq sample that was not treated with RNase H has high bisDRIP-seq score at the RPPH1 locus. On the other hand, there is a large decrease in bisDRIP-seq signal in the sample treated with RNase H. This suggests that bisDRIP-seq signal in the RPPH1 locus depends on the presence of an RNA-DNA hybrid.

    Techniques Used: Immunoprecipitation

    The strand asymmetry of bisDRIP-seq scores 3' of the transcription start site is sensitive to RNase H. ( A,B ) The strand asymmetry in bisDRIP-seq scores 3' of transcription start sites is not observed after RNase H treatment. RNA-DNA hybrids are core components of R-loops. If the strand asymmetry observed in metaplots of promoter regions is due to R-loops, than it should be selectively sensitive to RNase H. We therefore generated metaplots of bisDRIP-seq scores from two samples treated identically except that ( A ) received a vehicle treatment, while ( B ) was treated with RNase H. Metaplots were created by summing the bisDRIP-seq scores across active promoter regions (n = 15644) at each nucleotide position relative to the transcription start site. bisDRIP-seq scores were calculated separately for the nucleotide position on the non-template strand (blue) and template strand (orange). In the vehicle-treated sample, non-template bisDRIP-seq scores are higher than template bisDRIP-seq scores immediately 3' of the transcription start site. This replicates the result from Figure 3E . On the other hand, there is a complete loss of bisDRIP-seq score strand asymmetry after RNase H treatment in ( B ). This confirms that the asymmetry observed in Figure 3E is caused by R-loops.
    Figure Legend Snippet: The strand asymmetry of bisDRIP-seq scores 3' of the transcription start site is sensitive to RNase H. ( A,B ) The strand asymmetry in bisDRIP-seq scores 3' of transcription start sites is not observed after RNase H treatment. RNA-DNA hybrids are core components of R-loops. If the strand asymmetry observed in metaplots of promoter regions is due to R-loops, than it should be selectively sensitive to RNase H. We therefore generated metaplots of bisDRIP-seq scores from two samples treated identically except that ( A ) received a vehicle treatment, while ( B ) was treated with RNase H. Metaplots were created by summing the bisDRIP-seq scores across active promoter regions (n = 15644) at each nucleotide position relative to the transcription start site. bisDRIP-seq scores were calculated separately for the nucleotide position on the non-template strand (blue) and template strand (orange). In the vehicle-treated sample, non-template bisDRIP-seq scores are higher than template bisDRIP-seq scores immediately 3' of the transcription start site. This replicates the result from Figure 3E . On the other hand, there is a complete loss of bisDRIP-seq score strand asymmetry after RNase H treatment in ( B ). This confirms that the asymmetry observed in Figure 3E is caused by R-loops.

    Techniques Used: Generated

    R-loop signal in MALAT1 and NEAT1 is reduced by RNase H treatment. ( A,B ) bisDRIP-seq enrichment at ( A ) MALAT1 and ( B ) NEAT1 is lost after RNase H treatment and is not observed without RNA-DNA hybrid immunoprecipitation by S9.6 antibody. RNase H should selectively degrade the RNA component of R-loops. Thus, if bisDRIP-seq signal is caused by R-loops, RNase H treatment should reduce bisDRIP-seq signal. In the top panel, bisDRIP-seq scores from ‘input’ samples (mean of n = 2 samples) were mapped to each gene locus. bisDRIP-seq input samples were treated an identical manner to other bisDRIP-seq samples, except that no S9.6 antibody immunoprecipitation steps were performed. In the middle and lower plots, the bisDRIP-seq scores from two matched bisDRIP-seq experiments were mapped to each locus. The only difference between these experiments is that one sample was not treated with RNase H (middle plots, n = 1 sample), while the other sample was treated with RNase H (lower plots, n = 1 sample). At both loci, the sample that was not treated with RNase H replicated the bisDRIP-seq signal observed in standard bisDRIP-seq control experiments ( Figure 6A and B ). On the other hand, there is less bisDRIP-seq signal observed at each loci in both the input and RNase H-treated samples. These results suggest that RNA-DNA hybrids form in the MALAT1 and NEAT1 loci and that these RNA-DNA hybrids cause the high bisDRIP-seq scores observed at these loci. The location of the transcription start site is demarcated by ‘TSS’ and a dashed vertical line.
    Figure Legend Snippet: R-loop signal in MALAT1 and NEAT1 is reduced by RNase H treatment. ( A,B ) bisDRIP-seq enrichment at ( A ) MALAT1 and ( B ) NEAT1 is lost after RNase H treatment and is not observed without RNA-DNA hybrid immunoprecipitation by S9.6 antibody. RNase H should selectively degrade the RNA component of R-loops. Thus, if bisDRIP-seq signal is caused by R-loops, RNase H treatment should reduce bisDRIP-seq signal. In the top panel, bisDRIP-seq scores from ‘input’ samples (mean of n = 2 samples) were mapped to each gene locus. bisDRIP-seq input samples were treated an identical manner to other bisDRIP-seq samples, except that no S9.6 antibody immunoprecipitation steps were performed. In the middle and lower plots, the bisDRIP-seq scores from two matched bisDRIP-seq experiments were mapped to each locus. The only difference between these experiments is that one sample was not treated with RNase H (middle plots, n = 1 sample), while the other sample was treated with RNase H (lower plots, n = 1 sample). At both loci, the sample that was not treated with RNase H replicated the bisDRIP-seq signal observed in standard bisDRIP-seq control experiments ( Figure 6A and B ). On the other hand, there is less bisDRIP-seq signal observed at each loci in both the input and RNase H-treated samples. These results suggest that RNA-DNA hybrids form in the MALAT1 and NEAT1 loci and that these RNA-DNA hybrids cause the high bisDRIP-seq scores observed at these loci. The location of the transcription start site is demarcated by ‘TSS’ and a dashed vertical line.

    Techniques Used: Immunoprecipitation

    Histone genes are enriched in R-loops. ( A,B ) RNA polymerase II binding and RNA levels in the nucleus do not appear to explain the association between histone genes and R-loop formation. R-loop formation was calculated for each promoter region by taking the sum of bisDRIP-seq score strand asymmetry and the triptolide-sensitivity the non-template bisDRIP-seq scores. R-loop formation was then plotted against ( A ) nuclear RNA levels or ( B ) POLR2A (RNA polymerase II) chromatin immunoprecipitation (ChIP) signal for each promoter region. Promoter regions were plotted separately for non-histone genes (blue) and replication-dependent histone genes (orange). A linear regression for all genes was also plotted (blue). Both nuclear RNA levels and RNA polymerase II ChIP signal were weakly correlated with R-loop formation. Nevertheless, replication-dependent histone genes typically had higher R-loop formation scores than genes with similar promoter activity. This suggests that the nuclear RNA level of histones mRNAs in do not explain the high R-loop signal of these genes. It also suggests that the level of RNA polymerase II bound to histone genes does not explain the high R-loop signal observed at these genes. The bisDRIP-seq values in both plots were derived from the mean bisDRIP-seq scores of n = 13 control-treated samples and mean bisDRIP-seq scores of n = 2 triptolide-treated samples. In ( A ), we examined n = 1813 non-histone genes and n = 2 histone genes. In ( B ), we examined n = 2064 non-histone genes and n = 13 histone genes. TPKM refers to transcripts per kilobase million. The shaded areas around linear regression models represent 95% confidence intervals. ( C ) RNase H sensitivity of the bisDRIP-seq signal in histone genes. Metaplots were generated of histone gene non-template bisDRIP-seq score from three different bisDRIP-seq experiments. In the top plot, bisDRIP-seq scores were derived from ‘input’ samples (mean bisDRIP-seq score from n = 2 samples). These input samples received the entire bisDRIP-seq treatment except for the S9.6 immunoprecipitation enrichment steps. In the middle plot, bisDRIP-seq scores were derived from a bisDRIP-seq experiment that did not include an RNase H treatment (n = 1 sample). In the bottom plot, bisDRIP-seq scores were derived from an RNase H-treated bisDRIP-seq experiment (n = 1 sample). The bisDRIP-seq sample that was not treated with RNase H replicated the high non-template bisDRIP-seq scores observed in other bisDRIP-seq experiments at histone loci. On the other hand, almost no bisDRIP-seq signal was observed in either the input samples or in the bisDRIP-seq sample treated with RNase H. This suggests that the bisDRIP-seq signal observed at these gene loci is caused by R-loops. Transcription start sites are indicated by both ‘TSS’ and by dashed vertical lines. ( D,E ) bisDRIP-seq enrichment at ( D ) HIST1H2BG and ( E ) HIST1H1E is due to the presence of R-loops. In the middle and top plots, we compare the bisDRIP-seq from an RNase H treated sample (n = 1) and a bisDRIP-seq sample that was not treated with RNase H (n = 1), respectively. In both the middle and top plot, bisDRIP-seq scores from each experiment were mapped to the genomic region surrounding ( D ) HIST1H2BG and ( E ) HIST1H1E . At both loci, the bisDRIP-seq sample that was not treated with RNase H had similar bisDRIP-seq signal to our other bisDRIP-seq control experiments ( Figure 5D and E ). On the other hand, there is a clear reduction in bisDRIP-seq scores in the RNase H-treated sample. This decrease in signal after RNase H treatment suggests that RNA-DNA hybrids do form at these loci. In the bottom panel, the percentage of each nucleotide is plotted in 51 bp windows. There is no obvious relationship between nucleotide composition and bisDRIP-seq scores throughout these two genes.
    Figure Legend Snippet: Histone genes are enriched in R-loops. ( A,B ) RNA polymerase II binding and RNA levels in the nucleus do not appear to explain the association between histone genes and R-loop formation. R-loop formation was calculated for each promoter region by taking the sum of bisDRIP-seq score strand asymmetry and the triptolide-sensitivity the non-template bisDRIP-seq scores. R-loop formation was then plotted against ( A ) nuclear RNA levels or ( B ) POLR2A (RNA polymerase II) chromatin immunoprecipitation (ChIP) signal for each promoter region. Promoter regions were plotted separately for non-histone genes (blue) and replication-dependent histone genes (orange). A linear regression for all genes was also plotted (blue). Both nuclear RNA levels and RNA polymerase II ChIP signal were weakly correlated with R-loop formation. Nevertheless, replication-dependent histone genes typically had higher R-loop formation scores than genes with similar promoter activity. This suggests that the nuclear RNA level of histones mRNAs in do not explain the high R-loop signal of these genes. It also suggests that the level of RNA polymerase II bound to histone genes does not explain the high R-loop signal observed at these genes. The bisDRIP-seq values in both plots were derived from the mean bisDRIP-seq scores of n = 13 control-treated samples and mean bisDRIP-seq scores of n = 2 triptolide-treated samples. In ( A ), we examined n = 1813 non-histone genes and n = 2 histone genes. In ( B ), we examined n = 2064 non-histone genes and n = 13 histone genes. TPKM refers to transcripts per kilobase million. The shaded areas around linear regression models represent 95% confidence intervals. ( C ) RNase H sensitivity of the bisDRIP-seq signal in histone genes. Metaplots were generated of histone gene non-template bisDRIP-seq score from three different bisDRIP-seq experiments. In the top plot, bisDRIP-seq scores were derived from ‘input’ samples (mean bisDRIP-seq score from n = 2 samples). These input samples received the entire bisDRIP-seq treatment except for the S9.6 immunoprecipitation enrichment steps. In the middle plot, bisDRIP-seq scores were derived from a bisDRIP-seq experiment that did not include an RNase H treatment (n = 1 sample). In the bottom plot, bisDRIP-seq scores were derived from an RNase H-treated bisDRIP-seq experiment (n = 1 sample). The bisDRIP-seq sample that was not treated with RNase H replicated the high non-template bisDRIP-seq scores observed in other bisDRIP-seq experiments at histone loci. On the other hand, almost no bisDRIP-seq signal was observed in either the input samples or in the bisDRIP-seq sample treated with RNase H. This suggests that the bisDRIP-seq signal observed at these gene loci is caused by R-loops. Transcription start sites are indicated by both ‘TSS’ and by dashed vertical lines. ( D,E ) bisDRIP-seq enrichment at ( D ) HIST1H2BG and ( E ) HIST1H1E is due to the presence of R-loops. In the middle and top plots, we compare the bisDRIP-seq from an RNase H treated sample (n = 1) and a bisDRIP-seq sample that was not treated with RNase H (n = 1), respectively. In both the middle and top plot, bisDRIP-seq scores from each experiment were mapped to the genomic region surrounding ( D ) HIST1H2BG and ( E ) HIST1H1E . At both loci, the bisDRIP-seq sample that was not treated with RNase H had similar bisDRIP-seq signal to our other bisDRIP-seq control experiments ( Figure 5D and E ). On the other hand, there is a clear reduction in bisDRIP-seq scores in the RNase H-treated sample. This decrease in signal after RNase H treatment suggests that RNA-DNA hybrids do form at these loci. In the bottom panel, the percentage of each nucleotide is plotted in 51 bp windows. There is no obvious relationship between nucleotide composition and bisDRIP-seq scores throughout these two genes.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Activity Assay, Derivative Assay, Generated, Immunoprecipitation

    5) Product Images from "R‐loop formation during S phase is restricted by PrimPol‐mediated repriming"

    Article Title: R‐loop formation during S phase is restricted by PrimPol‐mediated repriming

    Journal: The EMBO Journal

    doi: 10.15252/embj.201899793

    R‐loops promote ( GAA ) 10 ‐dependent epigenetic instability of  BU ‐1 DRIP‐qPCR analysis reveals accumulation of R‐loops across the  BU‐1  locus in  primpol  cells. The DRIP signal was calculated as enrichment over RNase H‐treated samples and was normalised to −0.5 kb amplicon. The mean and SD for three biological replicates is presented. An unpaired  t ‐test was used to compare differences between matched amplicons in  primpol BU‐1A (GAA)10  and the other cell lines indicated. **** P  ≤ 0.0001, ns = not significant. DNA:RNA hybrids in  primpol BU‐1A (GAA)10 :Gg RNase H1 (see also   Appendix Fig S5 ) and  primpol BU‐1A (GAA)10 :hPrimPol. An unpaired  t ‐test on three biological replicates was used to compare differences to  primpol BU‐1A (GAA)10  for each matched amplicon. The bar represents the mean, and whiskers represent the SD. *** P  ≤ 0.001, ** P  ≤ 0.01, ns = not significant. Overexpression of chicken RNase H1 prevents (GAA) 10 ‐induced  BU‐1A  epigenetic instability in  primpol  cells. Fluctuation analysis was performed on three  primpol BU‐1A (GAA)10  clones. One‐way ANOVA was used to calculate the significance of differences in  BU‐1  instability between  primpol BU‐1A ΔG4  and other cell lines. **** P  ≤ 0.0001, ns = not significant. Diagram of the RNase H1 hybrid binding domain (HBD)–mCherry fusion and flow cytometry expression profiles of the construct in four clones. Western blots of the same four clones are shown in   Appendix Fig S6 . R‐loop stabilisation induces epigenetic instability of  BU‐1 . Bu‐1a fluctuation analysis of wild‐type cells expressing HBD‐mCherry. The scatter plots pool results from at least two different clones with matched HBD expression. Mean ± SD reported. **** P  ≤ 0.0001, *** P  ≤ 0.001, ns = not significant; one‐way ANOVA.
    Figure Legend Snippet: R‐loops promote ( GAA ) 10 ‐dependent epigenetic instability of BU ‐1 DRIP‐qPCR analysis reveals accumulation of R‐loops across the BU‐1 locus in primpol cells. The DRIP signal was calculated as enrichment over RNase H‐treated samples and was normalised to −0.5 kb amplicon. The mean and SD for three biological replicates is presented. An unpaired t ‐test was used to compare differences between matched amplicons in primpol BU‐1A (GAA)10 and the other cell lines indicated. **** P  ≤ 0.0001, ns = not significant. DNA:RNA hybrids in primpol BU‐1A (GAA)10 :Gg RNase H1 (see also Appendix Fig S5 ) and primpol BU‐1A (GAA)10 :hPrimPol. An unpaired t ‐test on three biological replicates was used to compare differences to primpol BU‐1A (GAA)10 for each matched amplicon. The bar represents the mean, and whiskers represent the SD. *** P  ≤ 0.001, ** P  ≤ 0.01, ns = not significant. Overexpression of chicken RNase H1 prevents (GAA) 10 ‐induced BU‐1A epigenetic instability in primpol cells. Fluctuation analysis was performed on three primpol BU‐1A (GAA)10 clones. One‐way ANOVA was used to calculate the significance of differences in BU‐1 instability between primpol BU‐1A ΔG4 and other cell lines. **** P  ≤ 0.0001, ns = not significant. Diagram of the RNase H1 hybrid binding domain (HBD)–mCherry fusion and flow cytometry expression profiles of the construct in four clones. Western blots of the same four clones are shown in Appendix Fig S6 . R‐loop stabilisation induces epigenetic instability of BU‐1 . Bu‐1a fluctuation analysis of wild‐type cells expressing HBD‐mCherry. The scatter plots pool results from at least two different clones with matched HBD expression. Mean ± SD reported. **** P  ≤ 0.0001, *** P  ≤ 0.001, ns = not significant; one‐way ANOVA.

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification, Over Expression, Binding Assay, Flow Cytometry, Expressing, Construct, Clone Assay, Western Blot

    PrimPol suppresses R‐loop formation in association with  DNA  secondary structure‐forming sequences across the  DT 40 genome Representative normalised DRIP‐seq data in two genes  COL22A1 , spanning over 200 kb, and  MYC . The locations of H‐DNA and G4 motifs are shown below the gene map. Wild type in blue;  primpol  in red. The corresponding RNase H‐treated samples are dashed. See   Materials and Methods  for further details of graphic generation. Metagene analysis of DRIP peak distribution in wild‐type and  primpol  DT40 cells compared with the distribution of the indicated features in the genome. DRIP peak heights in wild type and  primpol  DT40 normalised to  Drosophila  S2 spike‐in.  n  (wild type) = 41,445;  n  ( primpol ) = 48,648. Correlation of normalised DRIP peak heights in the overlapping peaks between wild type and  primpol . Blue line = 1:1 correlation; red line = linear regression through data. Correlation between H‐DNA‐forming sequences and all genes (white bar), and genes with DRIP peaks in wild‐type (blue) and  primpol  cells (red). Normalised DRIP peak heights in the genes identified as associating with H‐DNA. Correlation between G4 motifs ([G 3‐5 N 1‐7 ] 4 ) and all genes (white bar), and genes with DRIP peaks in wild‐type (blue) and  primpol  cells (red). Normalised DRIP peak heights in the genes identified as associating with G4 motifs ([G 3‐5 N 1‐7 ] 4 ). Data information:  P ‐values calculated with Mann–Whitney  U ‐test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1 st /3 rd  quartile + 1.5*IQR).
    Figure Legend Snippet: PrimPol suppresses R‐loop formation in association with DNA secondary structure‐forming sequences across the DT 40 genome Representative normalised DRIP‐seq data in two genes COL22A1 , spanning over 200 kb, and MYC . The locations of H‐DNA and G4 motifs are shown below the gene map. Wild type in blue; primpol in red. The corresponding RNase H‐treated samples are dashed. See Materials and Methods for further details of graphic generation. Metagene analysis of DRIP peak distribution in wild‐type and primpol DT40 cells compared with the distribution of the indicated features in the genome. DRIP peak heights in wild type and primpol DT40 normalised to Drosophila S2 spike‐in. n (wild type) = 41,445; n ( primpol ) = 48,648. Correlation of normalised DRIP peak heights in the overlapping peaks between wild type and primpol . Blue line = 1:1 correlation; red line = linear regression through data. Correlation between H‐DNA‐forming sequences and all genes (white bar), and genes with DRIP peaks in wild‐type (blue) and primpol cells (red). Normalised DRIP peak heights in the genes identified as associating with H‐DNA. Correlation between G4 motifs ([G 3‐5 N 1‐7 ] 4 ) and all genes (white bar), and genes with DRIP peaks in wild‐type (blue) and primpol cells (red). Normalised DRIP peak heights in the genes identified as associating with G4 motifs ([G 3‐5 N 1‐7 ] 4 ). Data information: P ‐values calculated with Mann–Whitney U ‐test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1 st /3 rd quartile + 1.5*IQR).

    Techniques Used: MANN-WHITNEY

    PrimPol suppresses R‐loop formation in association with DNA secondary structure‐forming sequences in BOBSC iPS cells Representative normalised RNA DIP‐seq data in the SKI locus. Wild type in blue; primpol in red. The locations of H‐DNA and G4 motifs are shown below the gene map. The corresponding RNase H‐treated samples are dashed. Since so little material was recovered following RNase H treatment, all samples were pooled prior to library generation. Metagene analysis of RNA‐DIP peak distribution in wild‐type and primpol BOBSC cells compared with the distribution of the indicated features in the genome. RNA DIP‐seq peak heights in wild‐type and primpol BOBSC cells normalised to a DT40 spike‐in. n (wild type) = 32,740; n ( primpol ) = 33,721. Correlation of normalised RNA DIP‐seq peak heights in the overlapping peaks between wild type and primpol . Blue line = 1:1 correlation; red line = linear regression through data. Correlation between H‐DNA‐forming sequences and all genes (white bar), and genes with DRIP peaks in wild‐type (blue) and primpol cells (red). Correlation between G4 motifs ([G 3‐5 N 1‐7 ] 4 ) and all genes (white bar), and genes with DRIP peaks in wild‐type (blue) and primpol cells (red). Normalised RNA DIP‐seq peak heights in the genes identified as associating with H‐DNA. Normalised RNA DIP‐seq peak heights in the genes identified as associating with G4 motifs ([G 3‐5 N 1‐7 ] 4 ). Data information: P ‐values calculated with Mann–Whitney U ‐test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1 st /3 rd quartile + 1.5*IQR).
    Figure Legend Snippet: PrimPol suppresses R‐loop formation in association with DNA secondary structure‐forming sequences in BOBSC iPS cells Representative normalised RNA DIP‐seq data in the SKI locus. Wild type in blue; primpol in red. The locations of H‐DNA and G4 motifs are shown below the gene map. The corresponding RNase H‐treated samples are dashed. Since so little material was recovered following RNase H treatment, all samples were pooled prior to library generation. Metagene analysis of RNA‐DIP peak distribution in wild‐type and primpol BOBSC cells compared with the distribution of the indicated features in the genome. RNA DIP‐seq peak heights in wild‐type and primpol BOBSC cells normalised to a DT40 spike‐in. n (wild type) = 32,740; n ( primpol ) = 33,721. Correlation of normalised RNA DIP‐seq peak heights in the overlapping peaks between wild type and primpol . Blue line = 1:1 correlation; red line = linear regression through data. Correlation between H‐DNA‐forming sequences and all genes (white bar), and genes with DRIP peaks in wild‐type (blue) and primpol cells (red). Correlation between G4 motifs ([G 3‐5 N 1‐7 ] 4 ) and all genes (white bar), and genes with DRIP peaks in wild‐type (blue) and primpol cells (red). Normalised RNA DIP‐seq peak heights in the genes identified as associating with H‐DNA. Normalised RNA DIP‐seq peak heights in the genes identified as associating with G4 motifs ([G 3‐5 N 1‐7 ] 4 ). Data information: P ‐values calculated with Mann–Whitney U ‐test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1 st /3 rd quartile + 1.5*IQR).

    Techniques Used: DNA Immunoprecipitation Sequencing, MANN-WHITNEY

    Loss of PrimPol leads to unscheduled S phase R‐loop formation Expression of geminin‐tagged chicken RNase H1‐YFP. Phases of the cell cycle were determined by staining DNA content in live cells by Hoechst 33342 ( X ‐axis). RNase H1‐YFP with or without the geminin degron protein is detected on the Y ‐axis. The RNase H1‐YFP‐geminin degron is degraded in G1. In contrast, RNase H1‐YFP levels remain stable irrespective of the phase of the cell cycle. 2n and 4n indicate the chromosome number before and after DNA replication. Bu‐1a fluctuation analysis of two independently derived primpol BU‐1A (GAA)10 :Gg RNase H1‐YFP‐geminin degron clones. Since the expression of the RNase H1‐YFP‐geminin degron construct is not stable (unlike the RNase H1‐YFP construct without the degron), Bu‐1a expression was assessed separately in the YFP +ve and YFP −ve cells within each clone. Statistical differences calculated the Kruskal–Wallis test. For all panels, at least 36 individual clones were analysed; mean ± SD reported. **** P ≤ 0.0001, *** P ≤ 0.001, ns = not significant. DRIP‐qPCR for R‐loops around the engineered +3.5 (GAA) 10 repeat in BU‐1 in different phases of the cell cycle. The location of the qPCR amplicons is indicated in the map at the top of the panel. The BU‐1 DRIP signal was normalised to −0.5 kb amplicon in G1‐arrested cells ( t = 0 h). See Fig EV4 for representative cell cycle synchronisation profiles. Black: wild type; red: primpol . Error bars = SD. **** P ≤ 0.0001, *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05. Workflow for the S9.6‐independent detection of newly synthesised R‐loops. See Materials and Methods for details. Validation of analysis of nascent DNA:RNA hybrid formation in BU‐1 locus. Enrichment of 4‐SU‐labelled RNA moiety of DNA:RNA hybrids was calculated relative to input in three independent asynchronous wild‐type (black) or primpol (red) cells, with or without exogenous RNase H treatment. Error bars = SD. ** P ≤ 0.01, * P ≤ 0.05, ns = not significant; unpaired t ‐test. Synchronisation and 4‐SU pulse labelling scheme to identify nascently formed DNA:RNA hybrids. Newly synthesised R‐loops in BU‐1 during S phase in wild type (black) and primpol (red). Error bars represent 1 SD of three biological repeats of the experiment. *** P ≤ 0.001, * P ≤ 0.05; unpaired t ‐test.
    Figure Legend Snippet: Loss of PrimPol leads to unscheduled S phase R‐loop formation Expression of geminin‐tagged chicken RNase H1‐YFP. Phases of the cell cycle were determined by staining DNA content in live cells by Hoechst 33342 ( X ‐axis). RNase H1‐YFP with or without the geminin degron protein is detected on the Y ‐axis. The RNase H1‐YFP‐geminin degron is degraded in G1. In contrast, RNase H1‐YFP levels remain stable irrespective of the phase of the cell cycle. 2n and 4n indicate the chromosome number before and after DNA replication. Bu‐1a fluctuation analysis of two independently derived primpol BU‐1A (GAA)10 :Gg RNase H1‐YFP‐geminin degron clones. Since the expression of the RNase H1‐YFP‐geminin degron construct is not stable (unlike the RNase H1‐YFP construct without the degron), Bu‐1a expression was assessed separately in the YFP +ve and YFP −ve cells within each clone. Statistical differences calculated the Kruskal–Wallis test. For all panels, at least 36 individual clones were analysed; mean ± SD reported. **** P ≤ 0.0001, *** P ≤ 0.001, ns = not significant. DRIP‐qPCR for R‐loops around the engineered +3.5 (GAA) 10 repeat in BU‐1 in different phases of the cell cycle. The location of the qPCR amplicons is indicated in the map at the top of the panel. The BU‐1 DRIP signal was normalised to −0.5 kb amplicon in G1‐arrested cells ( t = 0 h). See Fig EV4 for representative cell cycle synchronisation profiles. Black: wild type; red: primpol . Error bars = SD. **** P ≤ 0.0001, *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05. Workflow for the S9.6‐independent detection of newly synthesised R‐loops. See Materials and Methods for details. Validation of analysis of nascent DNA:RNA hybrid formation in BU‐1 locus. Enrichment of 4‐SU‐labelled RNA moiety of DNA:RNA hybrids was calculated relative to input in three independent asynchronous wild‐type (black) or primpol (red) cells, with or without exogenous RNase H treatment. Error bars = SD. ** P ≤ 0.01, * P ≤ 0.05, ns = not significant; unpaired t ‐test. Synchronisation and 4‐SU pulse labelling scheme to identify nascently formed DNA:RNA hybrids. Newly synthesised R‐loops in BU‐1 during S phase in wild type (black) and primpol (red). Error bars represent 1 SD of three biological repeats of the experiment. *** P ≤ 0.001, * P ≤ 0.05; unpaired t ‐test.

    Techniques Used: Expressing, Staining, Derivative Assay, Clone Assay, Construct, Real-time Polymerase Chain Reaction, Amplification

    6) Product Images from "Argonaute-based programmable RNase as a tool for cleavage of highly-structured RNA"

    Article Title: Argonaute-based programmable RNase as a tool for cleavage of highly-structured RNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky496

    Comparing cleavage activity of DISC and RNase H. ( A ) Schematic of matched and mismatched guide and target pairs used to target four TRs across the HIV-1 ΔDIS 5′UTR RNA. For each pair, the HIV-1 ΔDIS 5′UTR sequence is shown on top and the perfectly matched gDNA strand is shown on the bottom. Circle indicates target position complementary to the first position of the guide that does not pair due to structural restrains by the protein. Black arrowheads indicate cleavage site. Mismatches between the guide and target strands are indicated by a black box around the bases of the guide that are mutated to the bases shown below the box. ( B ) Quantified cleavage products from the assay using matched and mismatched guide and target pairs described in (A) are plotted with solid bars representing the average of three replicates and circles representing individual replicates. Cleavage that was not detectable by the assay is indicated by ‘nd’. ( C and D ) Comparing DISC (circles) and RNase H (triangles) cleavage of the unstructured 60-nt target (C) or of a structured 352-nt RNA target (D). Bars indicate average cleavage of three replicates.
    Figure Legend Snippet: Comparing cleavage activity of DISC and RNase H. ( A ) Schematic of matched and mismatched guide and target pairs used to target four TRs across the HIV-1 ΔDIS 5′UTR RNA. For each pair, the HIV-1 ΔDIS 5′UTR sequence is shown on top and the perfectly matched gDNA strand is shown on the bottom. Circle indicates target position complementary to the first position of the guide that does not pair due to structural restrains by the protein. Black arrowheads indicate cleavage site. Mismatches between the guide and target strands are indicated by a black box around the bases of the guide that are mutated to the bases shown below the box. ( B ) Quantified cleavage products from the assay using matched and mismatched guide and target pairs described in (A) are plotted with solid bars representing the average of three replicates and circles representing individual replicates. Cleavage that was not detectable by the assay is indicated by ‘nd’. ( C and D ) Comparing DISC (circles) and RNase H (triangles) cleavage of the unstructured 60-nt target (C) or of a structured 352-nt RNA target (D). Bars indicate average cleavage of three replicates.

    Techniques Used: Activity Assay, Sequencing

    7) Product Images from "Formation and Repair of Mismatches Containing Ribonucleotides and Oxidized Bases at Repeated DNA Sequences *"

    Article Title: Formation and Repair of Mismatches Containing Ribonucleotides and Oxidized Bases at Repeated DNA Sequences *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.679209

    BER and RER activity on complex mispairs containing oxidized bases and ribonucleotides. When POL β incorporates rCMP opposite 8-oxodG (dG*), RNase H2 is going to efficiently remove rC from the resulting 8-oxodG:rC mispair, whereas OGG1 repair of dG* is slightly reduced. Should 8-oxodG:rA arise after rAMP incorporation, RER will process the rA containing strand, whereas MUTYH-mediated BER will be inhibited. Possible interference on RER activity might occur by concurrent recognition of the lesion. In the likelihood of limiting MTH1 hydrolytic activity, 8-oxorGTP (rG*TP) might be used by POL β to produce rG*:dA mispairs. These substrates will be efficiently processed by MUTYH and RNase H2. Simultaneous BER and RER activities might lead to the formation of double strand breaks ( DSB ) or intermediate repair products of unknown reparability.
    Figure Legend Snippet: BER and RER activity on complex mispairs containing oxidized bases and ribonucleotides. When POL β incorporates rCMP opposite 8-oxodG (dG*), RNase H2 is going to efficiently remove rC from the resulting 8-oxodG:rC mispair, whereas OGG1 repair of dG* is slightly reduced. Should 8-oxodG:rA arise after rAMP incorporation, RER will process the rA containing strand, whereas MUTYH-mediated BER will be inhibited. Possible interference on RER activity might occur by concurrent recognition of the lesion. In the likelihood of limiting MTH1 hydrolytic activity, 8-oxorGTP (rG*TP) might be used by POL β to produce rG*:dA mispairs. These substrates will be efficiently processed by MUTYH and RNase H2. Simultaneous BER and RER activities might lead to the formation of double strand breaks ( DSB ) or intermediate repair products of unknown reparability.

    Techniques Used: Activity Assay

    8) Product Images from "The final step of 40S ribosomal subunit maturation is controlled by a dual key lock"

    Article Title: The final step of 40S ribosomal subunit maturation is controlled by a dual key lock

    Journal: bioRxiv

    doi: 10.1101/2020.07.29.226936

    Assessment of the size of the ITS1 in purified pre-40S particles. Comparison of RNAse H digestion and alkaline hydrolysis assays shows nucleotide resolution between bands. Left panel, RNase H digestion of rRNAs from pre-40S particles purified using a HASt tagged version of LTV1 as bait (HASt-LTV1). Right panel, alkaline hydrolysis (OH - ladder) of an RNA molecule containing the 18S-ITS1 sequence recognized by the 3’18S probe at its 5’ end (see Suppl. File 2). The samples were fractionated ob a 12% polyacrylamide gel and northern blot was probed with the 3’18S probe.
    Figure Legend Snippet: Assessment of the size of the ITS1 in purified pre-40S particles. Comparison of RNAse H digestion and alkaline hydrolysis assays shows nucleotide resolution between bands. Left panel, RNase H digestion of rRNAs from pre-40S particles purified using a HASt tagged version of LTV1 as bait (HASt-LTV1). Right panel, alkaline hydrolysis (OH - ladder) of an RNA molecule containing the 18S-ITS1 sequence recognized by the 3’18S probe at its 5’ end (see Suppl. File 2). The samples were fractionated ob a 12% polyacrylamide gel and northern blot was probed with the 3’18S probe.

    Techniques Used: Purification, Sequencing, Northern Blot

    RPS26 is required for rRNA cleavage at site 3 as well as NOB1 and DIM2 release. HEK cell lines expressing tagged version of LTV1, the catalytically inactive RIO1-D324A (RIO1 (kd)) or wild-type RIO1 (RIO1(wt)) were treated with scramble or RPS26 siRNAs for 48 h. a , RNase H assays were conducted as in Figure 4 on rRNAs of pre-40S particles purified with the mentioned StHA tagged bait, either from RPS26-depleted cells or from control cells (scramble siRNA). b , Signals corresponding to the 18S-E and 18S rRNA detected in (a) were quantified and represented as the 18S/18S-E ratio for the different pre-40S particles. Error bars, s.d. (n=3) c , Cell extracts and purified particles were analysed by Western Blot using the indicated antibodies. d , Bands corresponding to DIM2 and NOB1 (in the eluates) were quantified, corrected for pre-40S particle loading (using RPS19) and normalized to the control condition (set to 1). Error bars, s.d. (n=3).
    Figure Legend Snippet: RPS26 is required for rRNA cleavage at site 3 as well as NOB1 and DIM2 release. HEK cell lines expressing tagged version of LTV1, the catalytically inactive RIO1-D324A (RIO1 (kd)) or wild-type RIO1 (RIO1(wt)) were treated with scramble or RPS26 siRNAs for 48 h. a , RNase H assays were conducted as in Figure 4 on rRNAs of pre-40S particles purified with the mentioned StHA tagged bait, either from RPS26-depleted cells or from control cells (scramble siRNA). b , Signals corresponding to the 18S-E and 18S rRNA detected in (a) were quantified and represented as the 18S/18S-E ratio for the different pre-40S particles. Error bars, s.d. (n=3) c , Cell extracts and purified particles were analysed by Western Blot using the indicated antibodies. d , Bands corresponding to DIM2 and NOB1 (in the eluates) were quantified, corrected for pre-40S particle loading (using RPS19) and normalized to the control condition (set to 1). Error bars, s.d. (n=3).

    Techniques Used: Expressing, Purification, Western Blot

    Late RIO1(kd)StHA pre-40S particles contain a high proportion of mature 18S rRNA. a , Diagram representing steps of the pre-40S rRNA digestion by RNase H. b , RNase H assays were performed on RNAs extracted from pre-40S particles purified with the mentioned StHA tagged bait, and separated on a 12% polyacrylamide gel. The 18S rRNA and its precursors were revealed by the 3’18S radiolabeled probe. Bands are separated with single nucleotide resolution, as shown in Figure 4 – figure supplement 1 . c , Signals corresponding to the 18S-E and 18S rRNAs were quantified by phosphorimaging and represented by the 18S/18S-E ratio for the different purified pre-40S particles. The average of three independent experiments is shown, with the standard deviation indicated on top of the histogram.
    Figure Legend Snippet: Late RIO1(kd)StHA pre-40S particles contain a high proportion of mature 18S rRNA. a , Diagram representing steps of the pre-40S rRNA digestion by RNase H. b , RNase H assays were performed on RNAs extracted from pre-40S particles purified with the mentioned StHA tagged bait, and separated on a 12% polyacrylamide gel. The 18S rRNA and its precursors were revealed by the 3’18S radiolabeled probe. Bands are separated with single nucleotide resolution, as shown in Figure 4 – figure supplement 1 . c , Signals corresponding to the 18S-E and 18S rRNAs were quantified by phosphorimaging and represented by the 18S/18S-E ratio for the different purified pre-40S particles. The average of three independent experiments is shown, with the standard deviation indicated on top of the histogram.

    Techniques Used: Purification, Standard Deviation

    In vitro cleavage of the 18S-E pre-rRNA within pre-40S particles is stimulated by ATP addition. HEK cell lines expressing tagged versions of wild-type RIO1 (RIO1(wt)) or of the catalytically-inactive RIO1 (kd) were treated with scramble or RPS26 siRNAs for 48h to enrich particles in state A. Pre-40S particles were purified and incubated in the presence of 1 mM ATP, 1mM AMP-PNP, or without nucleotide (mock condition). a , RNAse H assays were performed on the RNAs extracted from the particles. b , The variation of cleavage efficiency with the different nucleotides is indicated by the 18S/18S-E ratio and normalized to the mock-treated sample (set to 1). The data correspond to five independent experiments. Analysis of the results with a unilateral paired Wilcoxon test (“sample greater than mock”) indicates p-values of 0.031 for samples RIO1(wt)-ATP, RIO1(wt)-AMP-PNP, RIO1(kd)-ATP, and 0.063 for RIO1(kd)-AMP-PNP. c , Superimposition of atomic models of State A and B reveals overlapping distances (grey lines) between atoms of Proline 351 from RIO1 (green) and of Arginine 247 from DIM2 (orange). RPS5, which seems to be repositioned upon association of RIO1 / dissociation of DIM2 from the pre-40S particle, is shown in violet (State A) or white (State B).
    Figure Legend Snippet: In vitro cleavage of the 18S-E pre-rRNA within pre-40S particles is stimulated by ATP addition. HEK cell lines expressing tagged versions of wild-type RIO1 (RIO1(wt)) or of the catalytically-inactive RIO1 (kd) were treated with scramble or RPS26 siRNAs for 48h to enrich particles in state A. Pre-40S particles were purified and incubated in the presence of 1 mM ATP, 1mM AMP-PNP, or without nucleotide (mock condition). a , RNAse H assays were performed on the RNAs extracted from the particles. b , The variation of cleavage efficiency with the different nucleotides is indicated by the 18S/18S-E ratio and normalized to the mock-treated sample (set to 1). The data correspond to five independent experiments. Analysis of the results with a unilateral paired Wilcoxon test (“sample greater than mock”) indicates p-values of 0.031 for samples RIO1(wt)-ATP, RIO1(wt)-AMP-PNP, RIO1(kd)-ATP, and 0.063 for RIO1(kd)-AMP-PNP. c , Superimposition of atomic models of State A and B reveals overlapping distances (grey lines) between atoms of Proline 351 from RIO1 (green) and of Arginine 247 from DIM2 (orange). RPS5, which seems to be repositioned upon association of RIO1 / dissociation of DIM2 from the pre-40S particle, is shown in violet (State A) or white (State B).

    Techniques Used: In Vitro, Expressing, Purification, Incubation

    9) Product Images from "Elongation Factor TFIIS Prevents Transcription Stress and R-Loop Accumulation to Maintain Genome Stability"

    Article Title: Elongation Factor TFIIS Prevents Transcription Stress and R-Loop Accumulation to Maintain Genome Stability

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2019.07.037

    TFIIS mut Expression Results in an Accumulation of R-Loops (A) RNA or DNA hybrid slot-blot of genomic DNA from TFIIS mut and parental cells, ±RNase H. S9.6 antibody was used to detect RNA or DNA hybrids (upper panel on right) with single-strand DNA antibody (bottom panel) as a loading control. Serial dilutions of genomic DNA (1/1 = 4 μg) were probed with S9.6 antibody for standards (left panel). (B) Fold enrichment in RNA or DNA hybrids compared with control (n = 3). Mean ± SEM (bars) values are shown. p values were determined by unpaired t test. (C and D) DRIP-qPCR analysis of R-loop induction at the SOX4 gene (C) and the SNRPN gene (D) (n = 3). Mean ± SEM (bars) values are shown. p values were determined by two-way ANOVA statistical test. (E) Left: schematic of idealized experiment. Radioactive label is denoted by red dot and the biotin tag on DNA with a black dot. The position of the first adenine in the transcript is also indicated. Right: R-loop detection by denaturing PAGE after addition of TFIIS proteins and RNase H to yeast TECs assembled in vitro . Ambion RNA size markers are indicated on the left for approximate RNA sizes. Positions of full-length product (FL), R-loops, and cleavage products are indicated on the right. (F) Left: experimental scheme; similar to that of (E) but involving purification via the biotin tag after RNase H digestion. Right: R-loop detection by denaturing PAGE after addition of TFIIS proteins and RNase H to yeast TECs assembled in vitro . Approximate RNA sizes RNA and position of R-loops are indicated on the right and next to relevant lanes. Asterisk-bar denotes irrelevant pausing sites of unknown origin, including IC1 and IC2. See Figure S5 for detailed schematic explanations.
    Figure Legend Snippet: TFIIS mut Expression Results in an Accumulation of R-Loops (A) RNA or DNA hybrid slot-blot of genomic DNA from TFIIS mut and parental cells, ±RNase H. S9.6 antibody was used to detect RNA or DNA hybrids (upper panel on right) with single-strand DNA antibody (bottom panel) as a loading control. Serial dilutions of genomic DNA (1/1 = 4 μg) were probed with S9.6 antibody for standards (left panel). (B) Fold enrichment in RNA or DNA hybrids compared with control (n = 3). Mean ± SEM (bars) values are shown. p values were determined by unpaired t test. (C and D) DRIP-qPCR analysis of R-loop induction at the SOX4 gene (C) and the SNRPN gene (D) (n = 3). Mean ± SEM (bars) values are shown. p values were determined by two-way ANOVA statistical test. (E) Left: schematic of idealized experiment. Radioactive label is denoted by red dot and the biotin tag on DNA with a black dot. The position of the first adenine in the transcript is also indicated. Right: R-loop detection by denaturing PAGE after addition of TFIIS proteins and RNase H to yeast TECs assembled in vitro . Ambion RNA size markers are indicated on the left for approximate RNA sizes. Positions of full-length product (FL), R-loops, and cleavage products are indicated on the right. (F) Left: experimental scheme; similar to that of (E) but involving purification via the biotin tag after RNase H digestion. Right: R-loop detection by denaturing PAGE after addition of TFIIS proteins and RNase H to yeast TECs assembled in vitro . Approximate RNA sizes RNA and position of R-loops are indicated on the right and next to relevant lanes. Asterisk-bar denotes irrelevant pausing sites of unknown origin, including IC1 and IC2. See Figure S5 for detailed schematic explanations.

    Techniques Used: Expressing, Dot Blot, Real-time Polymerase Chain Reaction, Polyacrylamide Gel Electrophoresis, In Vitro, Purification

    10) Product Images from "RNA/DNA Hybrid Interactome Identifies DXH9 as a Molecular Player in Transcriptional Termination and R-Loop-Associated DNA Damage"

    Article Title: RNA/DNA Hybrid Interactome Identifies DXH9 as a Molecular Player in Transcriptional Termination and R-Loop-Associated DNA Damage

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2018.04.025

    Validation of New RNA/DNA Hybrid Interactome Candidates (A) Workflow of RNA/DNA hybrid IP with RNase H digestion. (B and C) HeLa genomic DNA input was either treated (+) or not (−) with RNase H before enrichment for RNA/DNA hybrids with the S9.6 antibody. Genomic RNA/DNA hybrids were incubated with nuclear extracts depleted for RNA/DNA hybrids with RNase A, followed by S9.6 IP. RNA/DNA hybrid slot blot (B) and western blot of RNA/DNA hybrid IP, probed with indicated antibodies (C). (D–I) Genomic DNA from HeLa cells transfected with control (siCtrl) or indicated siRNAs was treated with RNase H. siTop1 (D), siDHX9 #1 (E), siWHSC1 (F), siSAFB2 (G), siDNA-PK (H), siPARP1 (I) were used. Top: RNA/DNA hybrid slot blot. Bottom: quantification of S9.6 signal. Values are normalized to the siCtrl and represent the means ± SEMs, n ≥ 3. See also Figure S3 .
    Figure Legend Snippet: Validation of New RNA/DNA Hybrid Interactome Candidates (A) Workflow of RNA/DNA hybrid IP with RNase H digestion. (B and C) HeLa genomic DNA input was either treated (+) or not (−) with RNase H before enrichment for RNA/DNA hybrids with the S9.6 antibody. Genomic RNA/DNA hybrids were incubated with nuclear extracts depleted for RNA/DNA hybrids with RNase A, followed by S9.6 IP. RNA/DNA hybrid slot blot (B) and western blot of RNA/DNA hybrid IP, probed with indicated antibodies (C). (D–I) Genomic DNA from HeLa cells transfected with control (siCtrl) or indicated siRNAs was treated with RNase H. siTop1 (D), siDHX9 #1 (E), siWHSC1 (F), siSAFB2 (G), siDNA-PK (H), siPARP1 (I) were used. Top: RNA/DNA hybrid slot blot. Bottom: quantification of S9.6 signal. Values are normalized to the siCtrl and represent the means ± SEMs, n ≥ 3. See also Figure S3 .

    Techniques Used: Incubation, Dot Blot, Western Blot, Transfection

    11) Product Images from "Translation initiation of alphavirus mRNA reveals new insights into the topology of the 48S initiation complex"

    Article Title: Translation initiation of alphavirus mRNA reveals new insights into the topology of the 48S initiation complex

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky071

    Effect of VIC oligo-4 on the interaction of eIF4A in the 48S complex with the base of the DLP structure. ( A ) 43S model showing the binding site of VIC oligo-4 to the ES6S C-D helix. VIC oligo-4 binding to purified rabbit 40S subunit was analyzed by RNAse H cleavage as described previously ( 21 ). The resulting 18S rRNA fragments were analyzed by electrophoresis in a 0.8% agarose gel. ( B ) Effect of VIC oligo-4 addition (10 μM) on translation programmed with SV DLP capsid and SV ΔDLP capsid mRNAs in RRL. Note that elimination of the DLP structure impaired AUG recognition, giving rise to aberrant products (gray arrowheads) that resulted from spurious initiation at downstream AUGs, as reported previously ( 24 ). ( C ) Crosslinking assays using SV DLPU1 mRNA in the presence of 10 μM of VIC oligo-4. Ribosomal fractions (P100) and post-ribosomal fractions (S100) were analyzed. The change in intensity of the eIF4A band was measured from three independent experiments; data are represented as the mean ± SEM. ( D ) Effect of VIC oligo-4 on 48S formation and eIF4A crosslinking. The experiment was performed as in Figure 1C ; as VIC oligo-4 reduced 48S formation, the volume of fraction 11 was adjusted to analyze an equivalent cpm. Arrows shows the direction of sedimentation.
    Figure Legend Snippet: Effect of VIC oligo-4 on the interaction of eIF4A in the 48S complex with the base of the DLP structure. ( A ) 43S model showing the binding site of VIC oligo-4 to the ES6S C-D helix. VIC oligo-4 binding to purified rabbit 40S subunit was analyzed by RNAse H cleavage as described previously ( 21 ). The resulting 18S rRNA fragments were analyzed by electrophoresis in a 0.8% agarose gel. ( B ) Effect of VIC oligo-4 addition (10 μM) on translation programmed with SV DLP capsid and SV ΔDLP capsid mRNAs in RRL. Note that elimination of the DLP structure impaired AUG recognition, giving rise to aberrant products (gray arrowheads) that resulted from spurious initiation at downstream AUGs, as reported previously ( 24 ). ( C ) Crosslinking assays using SV DLPU1 mRNA in the presence of 10 μM of VIC oligo-4. Ribosomal fractions (P100) and post-ribosomal fractions (S100) were analyzed. The change in intensity of the eIF4A band was measured from three independent experiments; data are represented as the mean ± SEM. ( D ) Effect of VIC oligo-4 on 48S formation and eIF4A crosslinking. The experiment was performed as in Figure 1C ; as VIC oligo-4 reduced 48S formation, the volume of fraction 11 was adjusted to analyze an equivalent cpm. Arrows shows the direction of sedimentation.

    Techniques Used: Binding Assay, Purification, Electrophoresis, Agarose Gel Electrophoresis, Sedimentation

    eIF4A activity within the 48S complex. ( A ) RNase H-mapping of RNA–RNA interactions between SV-DLP U1 and 18S rRNA. The analysis was carried out in the absence or presence of 1 μM hippuristanol, with identification of the resulting RNA fragments indicated. For clarity, a schematic diagram of the ES6S and h16–18 regions of rabbit 18S rRNA with the primers used for RNase H digestion is shown. The use of oligos 4 and 9 limited the region of 18S rRNA (509–830) where the crosslinkings concentrated. Bands corresponding to crosslinking of SV DLP U1 mRNA with the ES6S region (680–1863) and h16-h18 helices (1–662) were quantified by densitometry and expressed as a ratio. Data are the mean ± SEM from four independent experiments. ( B ) Effect of eIF4A inhibition on 48S toeprinting generated by SV-DLP U1 and SV-ΔDLP U1 mRNAs. Assays were carried out in the absence of presence of 1 μM of hippuristanol; positions of resulting primer extensions are annotated with respect to the A (+1) position of AUG, and they have been assigned with a precision of ±1 nt. The bottom shows a 2D structural model of the first 100 nt of 26S mRNA, showing the secondary structure derived from SHAPE analysis ( 21 ). Reactivity to SHAPE reagent (NMIA) is higher for unpaired nucleotides (red) and low for those involved in pairings (black). Stops corresponding to toeprints are marked with arrowheads. Quantification of toeprint ratios (17–19/23–25) in absence or presence of hippuristanol is shown from three independent experiments; data are the mean ± SEM.
    Figure Legend Snippet: eIF4A activity within the 48S complex. ( A ) RNase H-mapping of RNA–RNA interactions between SV-DLP U1 and 18S rRNA. The analysis was carried out in the absence or presence of 1 μM hippuristanol, with identification of the resulting RNA fragments indicated. For clarity, a schematic diagram of the ES6S and h16–18 regions of rabbit 18S rRNA with the primers used for RNase H digestion is shown. The use of oligos 4 and 9 limited the region of 18S rRNA (509–830) where the crosslinkings concentrated. Bands corresponding to crosslinking of SV DLP U1 mRNA with the ES6S region (680–1863) and h16-h18 helices (1–662) were quantified by densitometry and expressed as a ratio. Data are the mean ± SEM from four independent experiments. ( B ) Effect of eIF4A inhibition on 48S toeprinting generated by SV-DLP U1 and SV-ΔDLP U1 mRNAs. Assays were carried out in the absence of presence of 1 μM of hippuristanol; positions of resulting primer extensions are annotated with respect to the A (+1) position of AUG, and they have been assigned with a precision of ±1 nt. The bottom shows a 2D structural model of the first 100 nt of 26S mRNA, showing the secondary structure derived from SHAPE analysis ( 21 ). Reactivity to SHAPE reagent (NMIA) is higher for unpaired nucleotides (red) and low for those involved in pairings (black). Stops corresponding to toeprints are marked with arrowheads. Quantification of toeprint ratios (17–19/23–25) in absence or presence of hippuristanol is shown from three independent experiments; data are the mean ± SEM.

    Techniques Used: Activity Assay, Inhibition, Generated, Derivative Assay

    12) Product Images from "Isolation and genome-wide characterization of cellular DNA:RNA triplex structures"

    Article Title: Isolation and genome-wide characterization of cellular DNA:RNA triplex structures

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1305

    NEAT1 forms triplexes at numerous genomic sites. ( A ) NEAT1 profiles in TriplexRNA-seq (DNA-IP) (red) and nuclear RNA (blue) from HeLa S3 and U2OS cells with shaded TFR1 and TFR2. Minus (-) and plus (+) strands are shown. The position and sequence of NEAT1-TFR1 and -TFR2 are shown below. ( B ) EMSAs using 10 or 100 pmol of synthetic NEAT1 versions comprising TFR1 (40 or 52 nt) or TFR2 incubated with 0.25 pmol of double–stranded  32 P-labeled oligonucleotides which harbor sequences of NEAT1 target genes predicted from CHART-seq (  Supplementary Table S2 ). Reactions marked with an asterisk (*) were treated with 0.5 U RNase H. As a control, RNA without a putative TFR was used. Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). ( C ) Schematic depiction of the TFR-based capture assay. Biotinylated RNA oligos covering NEAT1-TFR1 and NEAT1-TFR2 were used to capture genomic DNA. ( D ) MEME motif analysis identifying consensus motifs in DNA captured by NEAT1-TFR1 (399 of top 500 peaks) and by NEAT1-TFR2 (500 of top 500 peaks ranked by peak  P -value). Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). ( E ) TDF analysis of the triplex-forming potential of NEAT1-TFR1 and NEAT1-TFR2 RNAs with top 500 TFR-associated and control DNA peaks (ranked by peak  P -value) compared to 500 randomized regions ( N  = 1000, colored grey).  P -values were obtained from one-tailed Mann–Whitney test. ( F ) Scheme presenting antisense oligo (ASO)-based capture of NEAT1-associated DNA. ( G ) Consensus motif in NEAT1-associated DNA sites (314 of top 500 peaks ranked by peak  P -value). ( H ) TDF analysis predicting the triplex-forming potential of NEAT1 on ASO-captured DNA regions. Significant TFRs along NEAT1 are shown in orange, the number of target sites (DBS) for each TFR in purple. For TFR- and ASO-based capture assays nucleic acids isolated from HeLa S3 chromatin were used.
    Figure Legend Snippet: NEAT1 forms triplexes at numerous genomic sites. ( A ) NEAT1 profiles in TriplexRNA-seq (DNA-IP) (red) and nuclear RNA (blue) from HeLa S3 and U2OS cells with shaded TFR1 and TFR2. Minus (-) and plus (+) strands are shown. The position and sequence of NEAT1-TFR1 and -TFR2 are shown below. ( B ) EMSAs using 10 or 100 pmol of synthetic NEAT1 versions comprising TFR1 (40 or 52 nt) or TFR2 incubated with 0.25 pmol of double–stranded 32 P-labeled oligonucleotides which harbor sequences of NEAT1 target genes predicted from CHART-seq ( Supplementary Table S2 ). Reactions marked with an asterisk (*) were treated with 0.5 U RNase H. As a control, RNA without a putative TFR was used. Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). ( C ) Schematic depiction of the TFR-based capture assay. Biotinylated RNA oligos covering NEAT1-TFR1 and NEAT1-TFR2 were used to capture genomic DNA. ( D ) MEME motif analysis identifying consensus motifs in DNA captured by NEAT1-TFR1 (399 of top 500 peaks) and by NEAT1-TFR2 (500 of top 500 peaks ranked by peak P -value). Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). ( E ) TDF analysis of the triplex-forming potential of NEAT1-TFR1 and NEAT1-TFR2 RNAs with top 500 TFR-associated and control DNA peaks (ranked by peak P -value) compared to 500 randomized regions ( N = 1000, colored grey). P -values were obtained from one-tailed Mann–Whitney test. ( F ) Scheme presenting antisense oligo (ASO)-based capture of NEAT1-associated DNA. ( G ) Consensus motif in NEAT1-associated DNA sites (314 of top 500 peaks ranked by peak P -value). ( H ) TDF analysis predicting the triplex-forming potential of NEAT1 on ASO-captured DNA regions. Significant TFRs along NEAT1 are shown in orange, the number of target sites (DBS) for each TFR in purple. For TFR- and ASO-based capture assays nucleic acids isolated from HeLa S3 chromatin were used.

    Techniques Used: Sequencing, Incubation, Labeling, One-tailed Test, MANN-WHITNEY, Allele-specific Oligonucleotide, Isolation

    Validation of triplex-forming RNA and DNAs. ( A ) TDF analysis predicting the potential of top 1000 enriched TriplexRNA (DNA-IP) regions (ranked by peak  P -value) to bind to active promoters defined by ChromHMM. Number of TFRs in RNA (per kilobase of RNA, left) and the number of putative DBSs at promoters (per kilobase of RNA, right) are shown. Boxplot borders are defined by the 1st and 3rd quantiles of the distributions, the middle line corresponds to the median value. The top whisker denotes the maximum value within the third quartile plus 1.5 times the interquartile range (bottom whisker is defined analogously). Dark gray dots represent outliers with values higher or lower than whiskers. Further box plots are based on the same definitions. ( B ) Motif analysis of triplexes formed between TriplexRNA (DNA-IP) and active promoters. The diagram depicts the fraction of antiparallel and parallel triplexes with the respective motif and nucleotide composition of TFRs in TriplexRNA. ( C ) TDF analysis comparing the triplex-forming potential of top 2000 TriplexDNA-seq regions with top 1000 TriplexRNA (DNA-IP) (ranked by peak  P -value). The number of putative DBSs (per kilobase of RNA) is shown. ( D ) Motif analysis of predicted triplexes formed between TriplexRNAs (DNA-IP) and TriplexDNA. The diagram depicts the fraction of antiparallel and parallel triplexes, with the respective motif and nucleotide composition of TFRs in TriplexRNA. ( E ) Box plot classifying triplex interactions between TriplexRNAs (DNA-IP) and TriplexDNA-seq regions as  cis  ( > 10 kb in the same chromosome) and  trans  (at different chromosomes) interactions, excluding underrepresented local interactions (within 10 kb distance). ( F ) EMSAs using 10 or 100 pmol of synthetic TriplexRNAs and 0.25 pmol of double–stranded  32 P-labeled oligonucleotides comprising target regions from TriplexDNA (  Supplementary Table S2 ). Reactions marked with an asterisk (*) were treated with 0.5 U RNase H. As a control (C), RNA without a putative TFR was used. Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). TriplexRNA-seq and TriplexDNA-seq data are from HeLa S3 cells. Adjusted  P -values
    Figure Legend Snippet: Validation of triplex-forming RNA and DNAs. ( A ) TDF analysis predicting the potential of top 1000 enriched TriplexRNA (DNA-IP) regions (ranked by peak P -value) to bind to active promoters defined by ChromHMM. Number of TFRs in RNA (per kilobase of RNA, left) and the number of putative DBSs at promoters (per kilobase of RNA, right) are shown. Boxplot borders are defined by the 1st and 3rd quantiles of the distributions, the middle line corresponds to the median value. The top whisker denotes the maximum value within the third quartile plus 1.5 times the interquartile range (bottom whisker is defined analogously). Dark gray dots represent outliers with values higher or lower than whiskers. Further box plots are based on the same definitions. ( B ) Motif analysis of triplexes formed between TriplexRNA (DNA-IP) and active promoters. The diagram depicts the fraction of antiparallel and parallel triplexes with the respective motif and nucleotide composition of TFRs in TriplexRNA. ( C ) TDF analysis comparing the triplex-forming potential of top 2000 TriplexDNA-seq regions with top 1000 TriplexRNA (DNA-IP) (ranked by peak P -value). The number of putative DBSs (per kilobase of RNA) is shown. ( D ) Motif analysis of predicted triplexes formed between TriplexRNAs (DNA-IP) and TriplexDNA. The diagram depicts the fraction of antiparallel and parallel triplexes, with the respective motif and nucleotide composition of TFRs in TriplexRNA. ( E ) Box plot classifying triplex interactions between TriplexRNAs (DNA-IP) and TriplexDNA-seq regions as cis ( > 10 kb in the same chromosome) and trans (at different chromosomes) interactions, excluding underrepresented local interactions (within 10 kb distance). ( F ) EMSAs using 10 or 100 pmol of synthetic TriplexRNAs and 0.25 pmol of double–stranded 32 P-labeled oligonucleotides comprising target regions from TriplexDNA ( Supplementary Table S2 ). Reactions marked with an asterisk (*) were treated with 0.5 U RNase H. As a control (C), RNA without a putative TFR was used. Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). TriplexRNA-seq and TriplexDNA-seq data are from HeLa S3 cells. Adjusted P -values

    Techniques Used: Whisker Assay, Labeling

    13) Product Images from "Isolation and genome-wide characterization of cellular DNA:RNA triplex structures"

    Article Title: Isolation and genome-wide characterization of cellular DNA:RNA triplex structures

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1305

    NEAT1 forms triplexes at numerous genomic sites. ( A ) NEAT1 profiles in TriplexRNA-seq (DNA-IP) (red) and nuclear RNA (blue) from HeLa S3 and U2OS cells with shaded TFR1 and TFR2. Minus (-) and plus (+) strands are shown. The position and sequence of NEAT1-TFR1 and -TFR2 are shown below. ( B ) EMSAs using 10 or 100 pmol of synthetic NEAT1 versions comprising TFR1 (40 or 52 nt) or TFR2 incubated with 0.25 pmol of double–stranded  32 P-labeled oligonucleotides which harbor sequences of NEAT1 target genes predicted from CHART-seq (  Supplementary Table S2 ). Reactions marked with an asterisk (*) were treated with 0.5 U RNase H. As a control, RNA without a putative TFR was used. Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). ( C ) Schematic depiction of the TFR-based capture assay. Biotinylated RNA oligos covering NEAT1-TFR1 and NEAT1-TFR2 were used to capture genomic DNA. ( D ) MEME motif analysis identifying consensus motifs in DNA captured by NEAT1-TFR1 (399 of top 500 peaks) and by NEAT1-TFR2 (500 of top 500 peaks ranked by peak  P -value). Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). ( E ) TDF analysis of the triplex-forming potential of NEAT1-TFR1 and NEAT1-TFR2 RNAs with top 500 TFR-associated and control DNA peaks (ranked by peak  P -value) compared to 500 randomized regions ( N  = 1000, colored grey).  P -values were obtained from one-tailed Mann–Whitney test. ( F ) Scheme presenting antisense oligo (ASO)-based capture of NEAT1-associated DNA. ( G ) Consensus motif in NEAT1-associated DNA sites (314 of top 500 peaks ranked by peak  P -value). ( H ) TDF analysis predicting the triplex-forming potential of NEAT1 on ASO-captured DNA regions. Significant TFRs along NEAT1 are shown in orange, the number of target sites (DBS) for each TFR in purple. For TFR- and ASO-based capture assays nucleic acids isolated from HeLa S3 chromatin were used.
    Figure Legend Snippet: NEAT1 forms triplexes at numerous genomic sites. ( A ) NEAT1 profiles in TriplexRNA-seq (DNA-IP) (red) and nuclear RNA (blue) from HeLa S3 and U2OS cells with shaded TFR1 and TFR2. Minus (-) and plus (+) strands are shown. The position and sequence of NEAT1-TFR1 and -TFR2 are shown below. ( B ) EMSAs using 10 or 100 pmol of synthetic NEAT1 versions comprising TFR1 (40 or 52 nt) or TFR2 incubated with 0.25 pmol of double–stranded 32 P-labeled oligonucleotides which harbor sequences of NEAT1 target genes predicted from CHART-seq ( Supplementary Table S2 ). Reactions marked with an asterisk (*) were treated with 0.5 U RNase H. As a control, RNA without a putative TFR was used. Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). ( C ) Schematic depiction of the TFR-based capture assay. Biotinylated RNA oligos covering NEAT1-TFR1 and NEAT1-TFR2 were used to capture genomic DNA. ( D ) MEME motif analysis identifying consensus motifs in DNA captured by NEAT1-TFR1 (399 of top 500 peaks) and by NEAT1-TFR2 (500 of top 500 peaks ranked by peak P -value). Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). ( E ) TDF analysis of the triplex-forming potential of NEAT1-TFR1 and NEAT1-TFR2 RNAs with top 500 TFR-associated and control DNA peaks (ranked by peak P -value) compared to 500 randomized regions ( N = 1000, colored grey). P -values were obtained from one-tailed Mann–Whitney test. ( F ) Scheme presenting antisense oligo (ASO)-based capture of NEAT1-associated DNA. ( G ) Consensus motif in NEAT1-associated DNA sites (314 of top 500 peaks ranked by peak P -value). ( H ) TDF analysis predicting the triplex-forming potential of NEAT1 on ASO-captured DNA regions. Significant TFRs along NEAT1 are shown in orange, the number of target sites (DBS) for each TFR in purple. For TFR- and ASO-based capture assays nucleic acids isolated from HeLa S3 chromatin were used.

    Techniques Used: Sequencing, Incubation, Labeling, One-tailed Test, MANN-WHITNEY, Allele-specific Oligonucleotide, Isolation

    Validation of triplex-forming RNA and DNAs. ( A ) TDF analysis predicting the potential of top 1000 enriched TriplexRNA (DNA-IP) regions (ranked by peak  P -value) to bind to active promoters defined by ChromHMM. Number of TFRs in RNA (per kilobase of RNA, left) and the number of putative DBSs at promoters (per kilobase of RNA, right) are shown. Boxplot borders are defined by the 1st and 3rd quantiles of the distributions, the middle line corresponds to the median value. The top whisker denotes the maximum value within the third quartile plus 1.5 times the interquartile range (bottom whisker is defined analogously). Dark gray dots represent outliers with values higher or lower than whiskers. Further box plots are based on the same definitions. ( B ) Motif analysis of triplexes formed between TriplexRNA (DNA-IP) and active promoters. The diagram depicts the fraction of antiparallel and parallel triplexes with the respective motif and nucleotide composition of TFRs in TriplexRNA. ( C ) TDF analysis comparing the triplex-forming potential of top 2000 TriplexDNA-seq regions with top 1000 TriplexRNA (DNA-IP) (ranked by peak  P -value). The number of putative DBSs (per kilobase of RNA) is shown. ( D ) Motif analysis of predicted triplexes formed between TriplexRNAs (DNA-IP) and TriplexDNA. The diagram depicts the fraction of antiparallel and parallel triplexes, with the respective motif and nucleotide composition of TFRs in TriplexRNA. ( E ) Box plot classifying triplex interactions between TriplexRNAs (DNA-IP) and TriplexDNA-seq regions as  cis  ( > 10 kb in the same chromosome) and  trans  (at different chromosomes) interactions, excluding underrepresented local interactions (within 10 kb distance). ( F ) EMSAs using 10 or 100 pmol of synthetic TriplexRNAs and 0.25 pmol of double–stranded  32 P-labeled oligonucleotides comprising target regions from TriplexDNA (  Supplementary Table S2 ). Reactions marked with an asterisk (*) were treated with 0.5 U RNase H. As a control (C), RNA without a putative TFR was used. Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). TriplexRNA-seq and TriplexDNA-seq data are from HeLa S3 cells. Adjusted  P -values
    Figure Legend Snippet: Validation of triplex-forming RNA and DNAs. ( A ) TDF analysis predicting the potential of top 1000 enriched TriplexRNA (DNA-IP) regions (ranked by peak P -value) to bind to active promoters defined by ChromHMM. Number of TFRs in RNA (per kilobase of RNA, left) and the number of putative DBSs at promoters (per kilobase of RNA, right) are shown. Boxplot borders are defined by the 1st and 3rd quantiles of the distributions, the middle line corresponds to the median value. The top whisker denotes the maximum value within the third quartile plus 1.5 times the interquartile range (bottom whisker is defined analogously). Dark gray dots represent outliers with values higher or lower than whiskers. Further box plots are based on the same definitions. ( B ) Motif analysis of triplexes formed between TriplexRNA (DNA-IP) and active promoters. The diagram depicts the fraction of antiparallel and parallel triplexes with the respective motif and nucleotide composition of TFRs in TriplexRNA. ( C ) TDF analysis comparing the triplex-forming potential of top 2000 TriplexDNA-seq regions with top 1000 TriplexRNA (DNA-IP) (ranked by peak P -value). The number of putative DBSs (per kilobase of RNA) is shown. ( D ) Motif analysis of predicted triplexes formed between TriplexRNAs (DNA-IP) and TriplexDNA. The diagram depicts the fraction of antiparallel and parallel triplexes, with the respective motif and nucleotide composition of TFRs in TriplexRNA. ( E ) Box plot classifying triplex interactions between TriplexRNAs (DNA-IP) and TriplexDNA-seq regions as cis ( > 10 kb in the same chromosome) and trans (at different chromosomes) interactions, excluding underrepresented local interactions (within 10 kb distance). ( F ) EMSAs using 10 or 100 pmol of synthetic TriplexRNAs and 0.25 pmol of double–stranded 32 P-labeled oligonucleotides comprising target regions from TriplexDNA ( Supplementary Table S2 ). Reactions marked with an asterisk (*) were treated with 0.5 U RNase H. As a control (C), RNA without a putative TFR was used. Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). TriplexRNA-seq and TriplexDNA-seq data are from HeLa S3 cells. Adjusted P -values

    Techniques Used: Whisker Assay, Labeling

    14) Product Images from "Functional Coupling of Last-Intron Splicing and 3?-End Processing to Transcription In Vitro: the Poly(A) Signal Couples to Splicing before Committing to Cleavage ▿Functional Coupling of Last-Intron Splicing and 3?-End Processing to Transcription In Vitro: the Poly(A) Signal Couples to Splicing before Committing to Cleavage ▿ †"

    Article Title: Functional Coupling of Last-Intron Splicing and 3?-End Processing to Transcription In Vitro: the Poly(A) Signal Couples to Splicing before Committing to Cleavage ▿Functional Coupling of Last-Intron Splicing and 3?-End Processing to Transcription In Vitro: the Poly(A) Signal Couples to Splicing before Committing to Cleavage ▿ †

    Journal:

    doi: 10.1128/MCB.01410-07

    RNase H cutting has much less effect on second-intron splicing when the SV40 late poly(A) signal defines the terminal exon. (A) This experiment was done as described in the legend to Fig. except that transcripts were postcut at the poly(A)
    Figure Legend Snippet: RNase H cutting has much less effect on second-intron splicing when the SV40 late poly(A) signal defines the terminal exon. (A) This experiment was done as described in the legend to Fig. except that transcripts were postcut at the poly(A)

    Techniques Used:

    15) Product Images from "Functional Coupling of Last-Intron Splicing and 3?-End Processing to Transcription In Vitro: the Poly(A) Signal Couples to Splicing before Committing to Cleavage ▿Functional Coupling of Last-Intron Splicing and 3?-End Processing to Transcription In Vitro: the Poly(A) Signal Couples to Splicing before Committing to Cleavage ▿ †"

    Article Title: Functional Coupling of Last-Intron Splicing and 3?-End Processing to Transcription In Vitro: the Poly(A) Signal Couples to Splicing before Committing to Cleavage ▿Functional Coupling of Last-Intron Splicing and 3?-End Processing to Transcription In Vitro: the Poly(A) Signal Couples to Splicing before Committing to Cleavage ▿ †

    Journal:

    doi: 10.1128/MCB.01410-07

    RNase H cutting has much less effect on second-intron splicing when the SV40 late poly(A) signal defines the terminal exon. (A) This experiment was done as described in the legend to Fig. except that transcripts were postcut at the poly(A)
    Figure Legend Snippet: RNase H cutting has much less effect on second-intron splicing when the SV40 late poly(A) signal defines the terminal exon. (A) This experiment was done as described in the legend to Fig. except that transcripts were postcut at the poly(A)

    Techniques Used:

    16) Product Images from "Functional Coupling of Last-Intron Splicing and 3?-End Processing to Transcription In Vitro: the Poly(A) Signal Couples to Splicing before Committing to Cleavage ▿Functional Coupling of Last-Intron Splicing and 3?-End Processing to Transcription In Vitro: the Poly(A) Signal Couples to Splicing before Committing to Cleavage ▿ †"

    Article Title: Functional Coupling of Last-Intron Splicing and 3?-End Processing to Transcription In Vitro: the Poly(A) Signal Couples to Splicing before Committing to Cleavage ▿Functional Coupling of Last-Intron Splicing and 3?-End Processing to Transcription In Vitro: the Poly(A) Signal Couples to Splicing before Committing to Cleavage ▿ †

    Journal:

    doi: 10.1128/MCB.01410-07

    RNase H cutting has much less effect on second-intron splicing when the SV40 late poly(A) signal defines the terminal exon. (A) This experiment was done as described in the legend to Fig. except that transcripts were postcut at the poly(A)
    Figure Legend Snippet: RNase H cutting has much less effect on second-intron splicing when the SV40 late poly(A) signal defines the terminal exon. (A) This experiment was done as described in the legend to Fig. except that transcripts were postcut at the poly(A)

    Techniques Used:

    17) Product Images from "Functional Coupling of Last-Intron Splicing and 3?-End Processing to Transcription In Vitro: the Poly(A) Signal Couples to Splicing before Committing to Cleavage ▿Functional Coupling of Last-Intron Splicing and 3?-End Processing to Transcription In Vitro: the Poly(A) Signal Couples to Splicing before Committing to Cleavage ▿ †"

    Article Title: Functional Coupling of Last-Intron Splicing and 3?-End Processing to Transcription In Vitro: the Poly(A) Signal Couples to Splicing before Committing to Cleavage ▿Functional Coupling of Last-Intron Splicing and 3?-End Processing to Transcription In Vitro: the Poly(A) Signal Couples to Splicing before Committing to Cleavage ▿ †

    Journal:

    doi: 10.1128/MCB.01410-07

    RNase H cutting has much less effect on second-intron splicing when the SV40 late poly(A) signal defines the terminal exon. (A) This experiment was done as described in the legend to Fig. except that transcripts were postcut at the poly(A)
    Figure Legend Snippet: RNase H cutting has much less effect on second-intron splicing when the SV40 late poly(A) signal defines the terminal exon. (A) This experiment was done as described in the legend to Fig. except that transcripts were postcut at the poly(A)

    Techniques Used:

    18) Product Images from "Functional Coupling of Last-Intron Splicing and 3?-End Processing to Transcription In Vitro: the Poly(A) Signal Couples to Splicing before Committing to Cleavage ▿Functional Coupling of Last-Intron Splicing and 3?-End Processing to Transcription In Vitro: the Poly(A) Signal Couples to Splicing before Committing to Cleavage ▿ †"

    Article Title: Functional Coupling of Last-Intron Splicing and 3?-End Processing to Transcription In Vitro: the Poly(A) Signal Couples to Splicing before Committing to Cleavage ▿Functional Coupling of Last-Intron Splicing and 3?-End Processing to Transcription In Vitro: the Poly(A) Signal Couples to Splicing before Committing to Cleavage ▿ †

    Journal:

    doi: 10.1128/MCB.01410-07

    RNase H cutting has much less effect on second-intron splicing when the SV40 late poly(A) signal defines the terminal exon. (A) This experiment was done as described in the legend to Fig. except that transcripts were postcut at the poly(A)
    Figure Legend Snippet: RNase H cutting has much less effect on second-intron splicing when the SV40 late poly(A) signal defines the terminal exon. (A) This experiment was done as described in the legend to Fig. except that transcripts were postcut at the poly(A)

    Techniques Used:

    19) Product Images from "Functional Coupling of Last-Intron Splicing and 3?-End Processing to Transcription In Vitro: the Poly(A) Signal Couples to Splicing before Committing to Cleavage ▿Functional Coupling of Last-Intron Splicing and 3?-End Processing to Transcription In Vitro: the Poly(A) Signal Couples to Splicing before Committing to Cleavage ▿ †"

    Article Title: Functional Coupling of Last-Intron Splicing and 3?-End Processing to Transcription In Vitro: the Poly(A) Signal Couples to Splicing before Committing to Cleavage ▿Functional Coupling of Last-Intron Splicing and 3?-End Processing to Transcription In Vitro: the Poly(A) Signal Couples to Splicing before Committing to Cleavage ▿ †

    Journal:

    doi: 10.1128/MCB.01410-07

    RNase H cutting has much less effect on second-intron splicing when the SV40 late poly(A) signal defines the terminal exon. (A) This experiment was done as described in the legend to Fig. except that transcripts were postcut at the poly(A)
    Figure Legend Snippet: RNase H cutting has much less effect on second-intron splicing when the SV40 late poly(A) signal defines the terminal exon. (A) This experiment was done as described in the legend to Fig. except that transcripts were postcut at the poly(A)

    Techniques Used:

    20) Product Images from "In vivo evidence that eIF3 stays bound to ribosomes elongating and terminating on short upstream ORFs to promote reinitiation"

    Article Title: In vivo evidence that eIF3 stays bound to ribosomes elongating and terminating on short upstream ORFs to promote reinitiation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx049

    Schematic representation of yeast in vivo RNA–protein Ni 2+ -pull down (RaP-NiP) assay using formaldehyde crosslinking. The basic scheme of the RaP-NiP is described in the form of a flowchart. Green and red balls represent 40S ribosomes and eIF3 complexes, respectively, grey balls stand for the Ni 2+ beads, and purple and blue balls depict some non-specific RNA binding proteins. Exponentially growing yeast cells were crosslinked with 1% formaldehyde. Crosslinking was stopped by adding glycine and the fixed cells were lysed using glass beads by rigorous vortexing. Pre-cleared whole cell extract (WCE) containing RaP-NiP mRNAs in protein-RNA complexes were selectively digested with RNase H using sequence specific custom-made oligos. The resulting specific mRNA segments were purified with the help of the His-tagged a/TIF32 subunit of yeast eIF3 or its mutant variants using the Ni-NTA sepharose beads. Thus isolated protein-RNA complexes were subsequently treated with Proteinase K, and the captured RNAs were further purified by hot phenol extraction, reverse transcribed and their amounts were then quantified by qRT-PCR. The schematic boxed on the right-hand side illustrates typical amounts of RNAse H digested RNA segments of REI-permissive uORF1 and REI-non-permissive uORF4 from the GCN4 mRNA leader co-purifying with eIF3, the typical ratio of which is ∼4:1.
    Figure Legend Snippet: Schematic representation of yeast in vivo RNA–protein Ni 2+ -pull down (RaP-NiP) assay using formaldehyde crosslinking. The basic scheme of the RaP-NiP is described in the form of a flowchart. Green and red balls represent 40S ribosomes and eIF3 complexes, respectively, grey balls stand for the Ni 2+ beads, and purple and blue balls depict some non-specific RNA binding proteins. Exponentially growing yeast cells were crosslinked with 1% formaldehyde. Crosslinking was stopped by adding glycine and the fixed cells were lysed using glass beads by rigorous vortexing. Pre-cleared whole cell extract (WCE) containing RaP-NiP mRNAs in protein-RNA complexes were selectively digested with RNase H using sequence specific custom-made oligos. The resulting specific mRNA segments were purified with the help of the His-tagged a/TIF32 subunit of yeast eIF3 or its mutant variants using the Ni-NTA sepharose beads. Thus isolated protein-RNA complexes were subsequently treated with Proteinase K, and the captured RNAs were further purified by hot phenol extraction, reverse transcribed and their amounts were then quantified by qRT-PCR. The schematic boxed on the right-hand side illustrates typical amounts of RNAse H digested RNA segments of REI-permissive uORF1 and REI-non-permissive uORF4 from the GCN4 mRNA leader co-purifying with eIF3, the typical ratio of which is ∼4:1.

    Techniques Used: In Vivo, RNA Binding Assay, Sequencing, Purification, Mutagenesis, Isolation, Quantitative RT-PCR

    eIF3 stabilizes the post-termination 40S complexes on stop codons of REI-permissive uORF1 and uORF2 from the GCN4 mRNA leader. ( A ) A schematic showing the wild type mRNA leader of the GCN4-lacZ fusion with colored bars indicating positions of individual RPEs of uORF1, as well as of uORF2 (color coding of all four uORFs reflects their REI-permissiveness (green) or -non-permissiveness (red)—for details see Supplementary Figures S1 and S2 ). The mRNA leader was divided into several segments (X, Y n and Z), where X and Z are present in all constructs shown in panel B and contain the RNase H cutting sites (indicated by scissors) and qRT-PCR primer binding sites (indicated by red arrows) at their 5΄ and 3΄ ends, respectively. The segment X is 151 bp in length (from position –229 to –79 relative to the uORF1 AUG start codon) and the segment Z encompasses the entire downstream sequence immediately following the uORF4 stop codon (i.e. from position +223 relative to the uORF1 AUG start codon downstream). The coordinates of all Y segments, by which the constructs in panel B differ and that are in each of them placed between the X and Z segments, are given at the top or bottom of the schematic. ( B ) Schematics showing individual uORF1–4 RaP-NiP constructs with corresponding Yn inserts of the same length for uORF1-only, 3-only and 4-only constructs (top three), and for the uORF2-only and uORF4_2-only constructs (bottom two). Black bars labeled as Y3’ represent composite 13+5 nt taken from the uORF3 3΄ UTR that were placed immediately behind the first 7 nt of the uORF4 3΄ UTR (i.e. behind the Y4 segment that ends exactly at the seventh nt of the uORF4 3΄ UTR) to keep the length of the uORF4-specific Y segment the same as that of uORF1. (We could not take the entire 3΄ UTR of uORF4 because in our set-up it is an integral part of the Z segment where the downstream qPCR primer base-pairs). ( C and D ) The RNase H-cleaved uORF1 segment specifically co-purifies with the His-tagged a/TIF32 subunit of eIF3 using the in vivo RaP-NiP. (C) The YMP1 ( gcn4Δ TIF32-His ) strain was introduced either with the uORF1-only RaP-NiP construct shown in panel B or an empty vector and the resulting transformants were pre-cultured in minimal media overnight, diluted to OD 600 ∼ 0.1 and further cultivated to OD 600 ∼1. The exponentially growing cells were then subjected to RaP-NiP as described in Materials and Methods and outlined in Figure 1 . Relative qPCR product levels (in %) of the Y1 segment of uORF1 recovered from each strain with standard deviations obtained from at least three independent experiments from three independent transformants (i.e. biological replicates) normalized to reference ACT1 mRNA as well as to total RNA levels are given with the values of uORF1-only set to 100 (asterisks indicate that P
    Figure Legend Snippet: eIF3 stabilizes the post-termination 40S complexes on stop codons of REI-permissive uORF1 and uORF2 from the GCN4 mRNA leader. ( A ) A schematic showing the wild type mRNA leader of the GCN4-lacZ fusion with colored bars indicating positions of individual RPEs of uORF1, as well as of uORF2 (color coding of all four uORFs reflects their REI-permissiveness (green) or -non-permissiveness (red)—for details see Supplementary Figures S1 and S2 ). The mRNA leader was divided into several segments (X, Y n and Z), where X and Z are present in all constructs shown in panel B and contain the RNase H cutting sites (indicated by scissors) and qRT-PCR primer binding sites (indicated by red arrows) at their 5΄ and 3΄ ends, respectively. The segment X is 151 bp in length (from position –229 to –79 relative to the uORF1 AUG start codon) and the segment Z encompasses the entire downstream sequence immediately following the uORF4 stop codon (i.e. from position +223 relative to the uORF1 AUG start codon downstream). The coordinates of all Y segments, by which the constructs in panel B differ and that are in each of them placed between the X and Z segments, are given at the top or bottom of the schematic. ( B ) Schematics showing individual uORF1–4 RaP-NiP constructs with corresponding Yn inserts of the same length for uORF1-only, 3-only and 4-only constructs (top three), and for the uORF2-only and uORF4_2-only constructs (bottom two). Black bars labeled as Y3’ represent composite 13+5 nt taken from the uORF3 3΄ UTR that were placed immediately behind the first 7 nt of the uORF4 3΄ UTR (i.e. behind the Y4 segment that ends exactly at the seventh nt of the uORF4 3΄ UTR) to keep the length of the uORF4-specific Y segment the same as that of uORF1. (We could not take the entire 3΄ UTR of uORF4 because in our set-up it is an integral part of the Z segment where the downstream qPCR primer base-pairs). ( C and D ) The RNase H-cleaved uORF1 segment specifically co-purifies with the His-tagged a/TIF32 subunit of eIF3 using the in vivo RaP-NiP. (C) The YMP1 ( gcn4Δ TIF32-His ) strain was introduced either with the uORF1-only RaP-NiP construct shown in panel B or an empty vector and the resulting transformants were pre-cultured in minimal media overnight, diluted to OD 600 ∼ 0.1 and further cultivated to OD 600 ∼1. The exponentially growing cells were then subjected to RaP-NiP as described in Materials and Methods and outlined in Figure 1 . Relative qPCR product levels (in %) of the Y1 segment of uORF1 recovered from each strain with standard deviations obtained from at least three independent experiments from three independent transformants (i.e. biological replicates) normalized to reference ACT1 mRNA as well as to total RNA levels are given with the values of uORF1-only set to 100 (asterisks indicate that P

    Techniques Used: Construct, Quantitative RT-PCR, Binding Assay, Sequencing, Labeling, Real-time Polymerase Chain Reaction, In Vivo, Plasmid Preparation, Cell Culture

    21) Product Images from "Direct and Rapid Detection of RNAs on a Novel RNA Microchip"

    Article Title: Direct and Rapid Detection of RNAs on a Novel RNA Microchip

    Journal: Chembiochem : a European journal of chemical biology

    doi: 10.1002/cbic.201000170

    RNA detection on the chip with single-nucleotide discrimination through RNase H digestion and Klenow extension at 60°C.
    Figure Legend Snippet: RNA detection on the chip with single-nucleotide discrimination through RNase H digestion and Klenow extension at 60°C.

    Techniques Used: RNA Detection, Chromatin Immunoprecipitation

    22) Product Images from "Characterizing the Coding Region Determinant-Binding Protein (CRD-BP)-Microphthalmia-associated Transcription Factor (MITF) mRNA interaction"

    Article Title: Characterizing the Coding Region Determinant-Binding Protein (CRD-BP)-Microphthalmia-associated Transcription Factor (MITF) mRNA interaction

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0171196

    Assessing the binding of MHO-1 and MHO-7 to MITF RNA and CRD-BP. (A) RNase protection assay to assess the binding of MHO-1 and MHO-7 antisense oligonucleotides to MITF RNA 1550–1740. 32 P-MITF RNA 1550–1740 was incubated with tRNA, MHO-1 or MHO-7 overnight at 42°C as described in Materials and Methods. The reactions were then subjected to RNase H treatment, precipitated, and ran on 8% denaturing polyacrylamide gel as shown. 32 P-MITF RNA fragments which were not digested by RNase H are shown by arrows. The 75 nts size KRAS RNA on lane 4 was used as a marker. The schematic diagram on the right illustrates the action of RNase H and the expected RNA fragments generated upon hybridization of MHO-1 and MHO-7 to 32 P-MITF RNA 1550–1740. (B) Fluorescence polarization assays to assess the binding of MHO-1 and MHO-7 to MITF RNA (left panel) and CRD-BP (right panel). Ten nM of fluorescein-labeled MHO-1 and MHO-7 AONs as well as fluorescein-labeled CD44 RNA were incubated with an increasing concentration of MITF RNA (0–1000 nM) (left panel) or CRD-BP (0–1000 nM) (right panel) as described in Materials and Methods. The error bars are S.E. and the results are representative of two separate experiments.
    Figure Legend Snippet: Assessing the binding of MHO-1 and MHO-7 to MITF RNA and CRD-BP. (A) RNase protection assay to assess the binding of MHO-1 and MHO-7 antisense oligonucleotides to MITF RNA 1550–1740. 32 P-MITF RNA 1550–1740 was incubated with tRNA, MHO-1 or MHO-7 overnight at 42°C as described in Materials and Methods. The reactions were then subjected to RNase H treatment, precipitated, and ran on 8% denaturing polyacrylamide gel as shown. 32 P-MITF RNA fragments which were not digested by RNase H are shown by arrows. The 75 nts size KRAS RNA on lane 4 was used as a marker. The schematic diagram on the right illustrates the action of RNase H and the expected RNA fragments generated upon hybridization of MHO-1 and MHO-7 to 32 P-MITF RNA 1550–1740. (B) Fluorescence polarization assays to assess the binding of MHO-1 and MHO-7 to MITF RNA (left panel) and CRD-BP (right panel). Ten nM of fluorescein-labeled MHO-1 and MHO-7 AONs as well as fluorescein-labeled CD44 RNA were incubated with an increasing concentration of MITF RNA (0–1000 nM) (left panel) or CRD-BP (0–1000 nM) (right panel) as described in Materials and Methods. The error bars are S.E. and the results are representative of two separate experiments.

    Techniques Used: Binding Assay, Rnase Protection Assay, Incubation, Marker, Generated, Hybridization, Fluorescence, Labeling, Concentration Assay

    23) Product Images from "Effects of Benzophenone-3 and Propylparaben on Estrogen Receptor–Dependent R-Loops and DNA Damage in Breast Epithelial Cells and Mice"

    Article Title: Effects of Benzophenone-3 and Propylparaben on Estrogen Receptor–Dependent R-Loops and DNA Damage in Breast Epithelial Cells and Mice

    Journal: Environmental Health Perspectives

    doi: 10.1289/EHP5221

    R-loop formation in T47D and MCF7 cells treated with 17 β -estradiol ( E 2 ), benzophenone-3 (BP-3), or propylparaben (PP) or vehicle with or without RNase H. (A) Immunostaining of R-loop with S9.6 antibody and DAPI in T47D cells treated with E 2 ( 10 nM ), BP-3 ( 5 μ M ) or PP ( 5 μ M ) without and with RNase H treatment following fixation. Scale bar = 20 μ M . (B) Quantification of the nuclear S9.6 intensity in T47D. (C) Quantification of nuclear S9.6 intensity in MCF-7. *** p
    Figure Legend Snippet: R-loop formation in T47D and MCF7 cells treated with 17 β -estradiol ( E 2 ), benzophenone-3 (BP-3), or propylparaben (PP) or vehicle with or without RNase H. (A) Immunostaining of R-loop with S9.6 antibody and DAPI in T47D cells treated with E 2 ( 10 nM ), BP-3 ( 5 μ M ) or PP ( 5 μ M ) without and with RNase H treatment following fixation. Scale bar = 20 μ M . (B) Quantification of the nuclear S9.6 intensity in T47D. (C) Quantification of nuclear S9.6 intensity in MCF-7. *** p

    Techniques Used: Immunostaining

    Characterization of 76N-Tert- ESR1 and R-loop formation in 76N-Tert- ESR1 following treatment with 17 β -estradiol ( E 2 ), benzophenone-3 (BP-3) or propylparaben (PP) with and without RNase H. (A) Map of pIN-ESR1 construct ESR1 insertion next to doxycycline(dox) inducible TRE 2 promoter. (B) Western blot ER α (upper panel) with MCF-7 as positive control (lane 1), 76N-Tert parental (lane 2), 76N-Tert- ESR1 without dox (lane 3), 76N-Tert- ESR1 with dox (lane 4), and 76N-Tert- ESR1 with dox and E 2 ( 10 nM ) treatment and β -actin as loading control (lower panel). (C) Immunostaining with S9.6 antibody and DAPI with 10 nM E 2 , 5 μ M BP-3, or 5 μ M PP treatment to parental 76N-Tert cells (upper panel), to 76N-Tert-ESR1 with dox induction (middle panel) without or with RNase H treatment (lower panel). Scale bar = 20 μ M . (D) Quantification of nuclear S9.6 intensity in (C). *** p
    Figure Legend Snippet: Characterization of 76N-Tert- ESR1 and R-loop formation in 76N-Tert- ESR1 following treatment with 17 β -estradiol ( E 2 ), benzophenone-3 (BP-3) or propylparaben (PP) with and without RNase H. (A) Map of pIN-ESR1 construct ESR1 insertion next to doxycycline(dox) inducible TRE 2 promoter. (B) Western blot ER α (upper panel) with MCF-7 as positive control (lane 1), 76N-Tert parental (lane 2), 76N-Tert- ESR1 without dox (lane 3), 76N-Tert- ESR1 with dox (lane 4), and 76N-Tert- ESR1 with dox and E 2 ( 10 nM ) treatment and β -actin as loading control (lower panel). (C) Immunostaining with S9.6 antibody and DAPI with 10 nM E 2 , 5 μ M BP-3, or 5 μ M PP treatment to parental 76N-Tert cells (upper panel), to 76N-Tert-ESR1 with dox induction (middle panel) without or with RNase H treatment (lower panel). Scale bar = 20 μ M . (D) Quantification of nuclear S9.6 intensity in (C). *** p

    Techniques Used: Construct, Western Blot, Positive Control, Immunostaining

    24) Product Images from "Effects of Benzophenone-3 and Propylparaben on Estrogen Receptor–Dependent R-Loops and DNA Damage in Breast Epithelial Cells and Mice"

    Article Title: Effects of Benzophenone-3 and Propylparaben on Estrogen Receptor–Dependent R-Loops and DNA Damage in Breast Epithelial Cells and Mice

    Journal: Environmental Health Perspectives

    doi: 10.1289/EHP5221

    R-loop formation in T47D and MCF7 cells treated with 17 β -estradiol ( E 2 ), benzophenone-3 (BP-3), or propylparaben (PP) or vehicle with or without RNase H. (A) Immunostaining of R-loop with S9.6 antibody and DAPI in T47D cells treated with E 2 ( 10 nM ), BP-3 ( 5 μ M ) or PP ( 5 μ M ) without and with RNase H treatment following fixation. Scale bar = 20 μ M . (B) Quantification of the nuclear S9.6 intensity in T47D. (C) Quantification of nuclear S9.6 intensity in MCF-7. *** p
    Figure Legend Snippet: R-loop formation in T47D and MCF7 cells treated with 17 β -estradiol ( E 2 ), benzophenone-3 (BP-3), or propylparaben (PP) or vehicle with or without RNase H. (A) Immunostaining of R-loop with S9.6 antibody and DAPI in T47D cells treated with E 2 ( 10 nM ), BP-3 ( 5 μ M ) or PP ( 5 μ M ) without and with RNase H treatment following fixation. Scale bar = 20 μ M . (B) Quantification of the nuclear S9.6 intensity in T47D. (C) Quantification of nuclear S9.6 intensity in MCF-7. *** p

    Techniques Used: Immunostaining

    Characterization of 76N-Tert- ESR1 and R-loop formation in 76N-Tert- ESR1 following treatment with 17 β -estradiol ( E 2 ), benzophenone-3 (BP-3) or propylparaben (PP) with and without RNase H. (A) Map of pIN-ESR1 construct ESR1 insertion next to doxycycline(dox) inducible TRE 2 promoter. (B) Western blot ER α (upper panel) with MCF-7 as positive control (lane 1), 76N-Tert parental (lane 2), 76N-Tert- ESR1 without dox (lane 3), 76N-Tert- ESR1 with dox (lane 4), and 76N-Tert- ESR1 with dox and E 2 ( 10 nM ) treatment and β -actin as loading control (lower panel). (C) Immunostaining with S9.6 antibody and DAPI with 10 nM E 2 , 5 μ M BP-3, or 5 μ M PP treatment to parental 76N-Tert cells (upper panel), to 76N-Tert-ESR1 with dox induction (middle panel) without or with RNase H treatment (lower panel). Scale bar = 20 μ M . (D) Quantification of nuclear S9.6 intensity in (C). *** p
    Figure Legend Snippet: Characterization of 76N-Tert- ESR1 and R-loop formation in 76N-Tert- ESR1 following treatment with 17 β -estradiol ( E 2 ), benzophenone-3 (BP-3) or propylparaben (PP) with and without RNase H. (A) Map of pIN-ESR1 construct ESR1 insertion next to doxycycline(dox) inducible TRE 2 promoter. (B) Western blot ER α (upper panel) with MCF-7 as positive control (lane 1), 76N-Tert parental (lane 2), 76N-Tert- ESR1 without dox (lane 3), 76N-Tert- ESR1 with dox (lane 4), and 76N-Tert- ESR1 with dox and E 2 ( 10 nM ) treatment and β -actin as loading control (lower panel). (C) Immunostaining with S9.6 antibody and DAPI with 10 nM E 2 , 5 μ M BP-3, or 5 μ M PP treatment to parental 76N-Tert cells (upper panel), to 76N-Tert-ESR1 with dox induction (middle panel) without or with RNase H treatment (lower panel). Scale bar = 20 μ M . (D) Quantification of nuclear S9.6 intensity in (C). *** p

    Techniques Used: Construct, Western Blot, Positive Control, Immunostaining

    25) Product Images from "RNA Polymerase III Regulates Cytosolic RNA:DNA Hybrids and Intracellular MicroRNA Expression *"

    Article Title: RNA Polymerase III Regulates Cytosolic RNA:DNA Hybrids and Intracellular MicroRNA Expression *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.636365

    RNA:DNA hybrids exist in the cytosol of human lung cancer cells. A , the human lung carcinoma cell line A549 was stained with the vital dsDNA-specific dye PicoGreen ( green ) at 10 μl/ml for 1 h and with the mitochondria-specific vital dye MitoTracker ( red ) at 100 n m  for 30 min. Samples shown in the  lower panels  were pretreated with 0.5 units/ml RNase H.  Scale bars  = 10 μm.  B  and  C , three-dimensional isosurface rendering ( B ) and quantification ( C ) of PicoGreen staining in the nucleus and cytosol of the images shown in  A . A one-tailed Wilcoxon test was performed.  Error bars  represent S.E. *,  p
    Figure Legend Snippet: RNA:DNA hybrids exist in the cytosol of human lung cancer cells. A , the human lung carcinoma cell line A549 was stained with the vital dsDNA-specific dye PicoGreen ( green ) at 10 μl/ml for 1 h and with the mitochondria-specific vital dye MitoTracker ( red ) at 100 n m for 30 min. Samples shown in the lower panels were pretreated with 0.5 units/ml RNase H. Scale bars = 10 μm. B and C , three-dimensional isosurface rendering ( B ) and quantification ( C ) of PicoGreen staining in the nucleus and cytosol of the images shown in A . A one-tailed Wilcoxon test was performed. Error bars represent S.E. *, p

    Techniques Used: Staining, One-tailed Test

    Cytosolic RNA:DNA hybrids interact with miRNA machinery proteins. A , cytosolic fractions of A549 cells were subjected to immunoprecipitation ( IP ) using RNA:DNA hybrid-specific antibody S9.6. Part of the cytosolic fraction was pretreated with 0.5 units/ml RNase H. Immunoprecipitated proteins were detected by SDS-PAGE and silver staining. The indicated bands were analyzed by mass spectrometry.  C , cytosolic;  N , nuclear.  B  and  C , A549 cells were treated with DMSO or 10 μ m  Ara-C for 15 h and harvested for cell fractionation after fixation. Cytosolic fractions were subjected to immunoprecipitation with RNA:DNA hybrid-specific antibody S9.6. Immunoblot analysis was carried out on immunoprecipitated proteins probed with antibodies specific for DDX17 ( B ) and AGO2 ( C ).
    Figure Legend Snippet: Cytosolic RNA:DNA hybrids interact with miRNA machinery proteins. A , cytosolic fractions of A549 cells were subjected to immunoprecipitation ( IP ) using RNA:DNA hybrid-specific antibody S9.6. Part of the cytosolic fraction was pretreated with 0.5 units/ml RNase H. Immunoprecipitated proteins were detected by SDS-PAGE and silver staining. The indicated bands were analyzed by mass spectrometry. C , cytosolic; N , nuclear. B and C , A549 cells were treated with DMSO or 10 μ m Ara-C for 15 h and harvested for cell fractionation after fixation. Cytosolic fractions were subjected to immunoprecipitation with RNA:DNA hybrid-specific antibody S9.6. Immunoblot analysis was carried out on immunoprecipitated proteins probed with antibodies specific for DDX17 ( B ) and AGO2 ( C ).

    Techniques Used: Immunoprecipitation, SDS Page, Silver Staining, Mass Spectrometry, Acetylene Reduction Assay, Cell Fractionation

    26) Product Images from "Establishment of an in vitro trans-splicing system in Trypanosoma brucei that requires endogenous spliced leader RNA"

    Article Title: Establishment of an in vitro trans-splicing system in Trypanosoma brucei that requires endogenous spliced leader RNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq065

    RNaseH digestion of U2 or U6 snRNAs inhibits in vitro trans- splicing. ( A ) ( a ) Whole-cell extract was incubated for 1 h on ice with oligonucleotides complementary to U3, U2 or U6 snRNAs, in the presence of 1U RNase H, as described in ‘Materials and Methods’ section. The RNA from the different reactions was analyzed by an RNase protection assay. RNA was separated on a 6% sequencing gel. Lane contents are as follows: 1, Probe (500 c.p.m.); 2, RNA from in vitro trans- splicing extract (control); 3, extract was subjected to RNase H cleavage with U3-specific oligonucleotide; 4, extract was subjected to RNase H cleavage with U2-specific oligonucleotide; 5, extract was subjected to RNase H cleavage with U6-specific oligonucleotide. M-DNA marker, labeled pBR322 DNA MspI digest. ( b ) Quantitation of the effect on the production of the trans -spliced product; data represent three independent experiments. ( B ) Primer extension to determine the amount of U2 snRNA after RNase H treatment. Primer extension was performed on RNase H-oligonucleotide cleaved extracts using U2 specific primer (listed in S-1). The identity of the antisense oligonucleotides used for cleavage is indicated . ( C ) Primer extension to determine the amount of U6 snRNA after RNase H treatment. Primer extension was performed on RNase H-oligonucleotide cleaved extracts using U6 specific primer (listed in S-1). The identity of the antisense oligonucleotides used for cleavage is indicated.
    Figure Legend Snippet: RNaseH digestion of U2 or U6 snRNAs inhibits in vitro trans- splicing. ( A ) ( a ) Whole-cell extract was incubated for 1 h on ice with oligonucleotides complementary to U3, U2 or U6 snRNAs, in the presence of 1U RNase H, as described in ‘Materials and Methods’ section. The RNA from the different reactions was analyzed by an RNase protection assay. RNA was separated on a 6% sequencing gel. Lane contents are as follows: 1, Probe (500 c.p.m.); 2, RNA from in vitro trans- splicing extract (control); 3, extract was subjected to RNase H cleavage with U3-specific oligonucleotide; 4, extract was subjected to RNase H cleavage with U2-specific oligonucleotide; 5, extract was subjected to RNase H cleavage with U6-specific oligonucleotide. M-DNA marker, labeled pBR322 DNA MspI digest. ( b ) Quantitation of the effect on the production of the trans -spliced product; data represent three independent experiments. ( B ) Primer extension to determine the amount of U2 snRNA after RNase H treatment. Primer extension was performed on RNase H-oligonucleotide cleaved extracts using U2 specific primer (listed in S-1). The identity of the antisense oligonucleotides used for cleavage is indicated . ( C ) Primer extension to determine the amount of U6 snRNA after RNase H treatment. Primer extension was performed on RNase H-oligonucleotide cleaved extracts using U6 specific primer (listed in S-1). The identity of the antisense oligonucleotides used for cleavage is indicated.

    Techniques Used: In Vitro, Incubation, Rnase Protection Assay, Sequencing, Marker, Labeling, Quantitation Assay

    27) Product Images from "Antisense-mediated affinity purification of dengue virus ribonucleoprotein complexes from infected cells"

    Article Title: Antisense-mediated affinity purification of dengue virus ribonucleoprotein complexes from infected cells

    Journal: Methods (San Diego, Calif.)

    doi: 10.1016/j.ymeth.2015.08.008

    RNase H mapping antisense oligonucleotide positions A) Schematic of the RNase H mapping assay. Target RNA is incubated with complementary antisense DNA oligonucleotides and RNase H. If hybridization occurs, RNase H will cleave the RNA involved in the RNA:DNA hybrid. Cleavage is measured using RT-qPCR. B) Infected cells were cross-linked, lysed, and incubated with candidate antisense DNA oligonucleotides and RNase H. Purified RNA from mock cross-linked cells was used as a positive control for RNase H cleavage. RNA was purified from RNase H reactions and cleavage assessed by RT-qPCR using qPCR primers that amplify across the oligonucleotide hybridization site. Data is represented as the amount of RNA remaining relative to the no oligo control.
    Figure Legend Snippet: RNase H mapping antisense oligonucleotide positions A) Schematic of the RNase H mapping assay. Target RNA is incubated with complementary antisense DNA oligonucleotides and RNase H. If hybridization occurs, RNase H will cleave the RNA involved in the RNA:DNA hybrid. Cleavage is measured using RT-qPCR. B) Infected cells were cross-linked, lysed, and incubated with candidate antisense DNA oligonucleotides and RNase H. Purified RNA from mock cross-linked cells was used as a positive control for RNase H cleavage. RNA was purified from RNase H reactions and cleavage assessed by RT-qPCR using qPCR primers that amplify across the oligonucleotide hybridization site. Data is represented as the amount of RNA remaining relative to the no oligo control.

    Techniques Used: Mapping Assay, Incubation, Hybridization, Quantitative RT-PCR, Infection, Purification, Positive Control, Real-time Polymerase Chain Reaction

    28) Product Images from "Improvement of RNA secondary structure prediction using RNase H cleavage and randomized oligonucleotides"

    Article Title: Improvement of RNA secondary structure prediction using RNase H cleavage and randomized oligonucleotides

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp587

    Phylogenetic secondary structure, the predicted lowest free energy secondary structure, and the four suboptimal structures of E. coli 5S rRNA. Loops A–E are labeled in the phylogenetic structure. Base-paired regions that are predicted correctly in the suboptimal structures are shaded. Nucleotides cleaved by RNase H cleavage are circled. The lowest free energy structure only has 27% of the base pairs present in the phylogenetic structure.
    Figure Legend Snippet: Phylogenetic secondary structure, the predicted lowest free energy secondary structure, and the four suboptimal structures of E. coli 5S rRNA. Loops A–E are labeled in the phylogenetic structure. Base-paired regions that are predicted correctly in the suboptimal structures are shaded. Nucleotides cleaved by RNase H cleavage are circled. The lowest free energy structure only has 27% of the base pairs present in the phylogenetic structure.

    Techniques Used: Labeling

    Representative gel autoradiogram of RNase H cleavage experiments to identify single-stranded regions in yeast tRNA Phe with randomized 5-mer oligonucleotides.
    Figure Legend Snippet: Representative gel autoradiogram of RNase H cleavage experiments to identify single-stranded regions in yeast tRNA Phe with randomized 5-mer oligonucleotides.

    Techniques Used:

    Schematic of the general method used in this study. Randomized DNA oligonucleotides are incubated with an RNA of interest. Only DNAs complementary to single-stranded regions bind, inducing RNase H cleavage of the RNA strand. Nucleotides which are subject to RNase H cleavage are used as single-stranded constraints in RNA secondary structure prediction.
    Figure Legend Snippet: Schematic of the general method used in this study. Randomized DNA oligonucleotides are incubated with an RNA of interest. Only DNAs complementary to single-stranded regions bind, inducing RNase H cleavage of the RNA strand. Nucleotides which are subject to RNase H cleavage are used as single-stranded constraints in RNA secondary structure prediction.

    Techniques Used: Incubation

    Representative gel autoradiogram of RNase H cleavage experiments to identify single-stranded regions in E. coli 5S rRNA. The numbers above the lanes indicate to which nucleotides in the RNA the oligonucleotide probe is complementary.
    Figure Legend Snippet: Representative gel autoradiogram of RNase H cleavage experiments to identify single-stranded regions in E. coli 5S rRNA. The numbers above the lanes indicate to which nucleotides in the RNA the oligonucleotide probe is complementary.

    Techniques Used:

    Phylogenetic secondary structure and three predicted suboptimal structures of yeast tRNA Phe . The lowest free energy structure has 95% of the base pairs predicted correctly. Base paired regions that are predicted correctly in the suboptimal structures are shaded. Nucleotides cleaved by RNase H after 1 or 2 h incubation are circled. D denotes dihydrouracil while Y denotes wybutosine.
    Figure Legend Snippet: Phylogenetic secondary structure and three predicted suboptimal structures of yeast tRNA Phe . The lowest free energy structure has 95% of the base pairs predicted correctly. Base paired regions that are predicted correctly in the suboptimal structures are shaded. Nucleotides cleaved by RNase H after 1 or 2 h incubation are circled. D denotes dihydrouracil while Y denotes wybutosine.

    Techniques Used: Incubation

    29) Product Images from "Engineering circular RNA for potent and stable translation in eukaryotic cells"

    Article Title: Engineering circular RNA for potent and stable translation in eukaryotic cells

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05096-6

    Permuted intron-exon splicing and addition of homology arms.  a  Schematic diagram showing permuted intron-exon construct design and mechanism of splicing. The group I catalytic intron of the T4 phage Td gene is bisected in such a way to preserve structural elements critical for ribozyme folding. Exon fragment 2 is then ligated upstream of exon fragment 1, and a coding region roughly 1.1 kb in length is inserted between the exon-exon junction. During splicing, the 3′ hydroxyl group of a guanosine nucleotide engages in a transesterification reaction at the 5′ splice site. The 5′ intron half is excised, and the freed hydroxyl group at the end of the intermediate engages in a second transesterification at the 3′ splice site, resulting in circularization of the intervening region and excision of the 3′ intron.  b  RNAFold predictions of precursor RNA secondary structure for homology arm design. Colors denote base pairing probability, with red indicating higher probability. Without homology arms, no base pairing is predicted to occur between the ends of the precursor molecule.  c  Agarose gel demonstrating the effect of homology arms on splicing. Putative circRNA runs at a higher molecular weight than heavier precursor RNA, as indicated. (−): no homology arms. Weak: weak homology arms, 9 nt. Strong: strong homology arms, 19 nt.  d  Agarose gel confirmation of precursor RNA circularization. C: precursor RNA (with strong homology arms) subjected to circularization conditions. C + R: Lane C, digested with RNase R. C + R + H: Lane C + R, digested with oligonucleotide-guided RNase H. U: precursor RNA not subjected to circularization conditions. U + H: Lane U, digested with oligonucleotide-guided RNase H.  e  Sanger sequencing output of RT-PCR across the splice junction of the sample depicted in lane C + R from ( d )
    Figure Legend Snippet: Permuted intron-exon splicing and addition of homology arms. a Schematic diagram showing permuted intron-exon construct design and mechanism of splicing. The group I catalytic intron of the T4 phage Td gene is bisected in such a way to preserve structural elements critical for ribozyme folding. Exon fragment 2 is then ligated upstream of exon fragment 1, and a coding region roughly 1.1 kb in length is inserted between the exon-exon junction. During splicing, the 3′ hydroxyl group of a guanosine nucleotide engages in a transesterification reaction at the 5′ splice site. The 5′ intron half is excised, and the freed hydroxyl group at the end of the intermediate engages in a second transesterification at the 3′ splice site, resulting in circularization of the intervening region and excision of the 3′ intron. b RNAFold predictions of precursor RNA secondary structure for homology arm design. Colors denote base pairing probability, with red indicating higher probability. Without homology arms, no base pairing is predicted to occur between the ends of the precursor molecule. c Agarose gel demonstrating the effect of homology arms on splicing. Putative circRNA runs at a higher molecular weight than heavier precursor RNA, as indicated. (−): no homology arms. Weak: weak homology arms, 9 nt. Strong: strong homology arms, 19 nt. d Agarose gel confirmation of precursor RNA circularization. C: precursor RNA (with strong homology arms) subjected to circularization conditions. C + R: Lane C, digested with RNase R. C + R + H: Lane C + R, digested with oligonucleotide-guided RNase H. U: precursor RNA not subjected to circularization conditions. U + H: Lane U, digested with oligonucleotide-guided RNase H. e Sanger sequencing output of RT-PCR across the splice junction of the sample depicted in lane C + R from ( d )

    Techniques Used: Construct, Agarose Gel Electrophoresis, Molecular Weight, Sequencing, Reverse Transcription Polymerase Chain Reaction

    30) Product Images from "Strong transcription blockage mediated by R-loop formation within a G-rich homopurine–homopyrimidine sequence localized in the vicinity of the promoter"

    Article Title: Strong transcription blockage mediated by R-loop formation within a G-rich homopurine–homopyrimidine sequence localized in the vicinity of the promoter

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx403

    Effect of RNase H upon transcription. Substrates containing the G-rich sequence were used in these experiments. See the Results section for description of the experiment. ( A ) Gel image. ( B ) Quantitation of the results. All run-off signals are normalized to the signal for promoter–distal substrate transcribed without RNase H.
    Figure Legend Snippet: Effect of RNase H upon transcription. Substrates containing the G-rich sequence were used in these experiments. See the Results section for description of the experiment. ( A ) Gel image. ( B ) Quantitation of the results. All run-off signals are normalized to the signal for promoter–distal substrate transcribed without RNase H.

    Techniques Used: Sequencing, Quantitation Assay

    Model for transcription blockage by R-loop formation in the vicinity of the promoter. The R-loop-prone (G-rich) DNA sequence is shown in turquoise, the rest of DNA is shown in gray, transcript from the R-loop-prone sequence is shown in dark blue, the rest of RNA is shown in black, a bent arrow indicates the transcription start site. RNA polymerase (RNAP) is shown as a gray circle. During transcription, an R-loop is formed with a certain probability p , while transcription proceeds without R-loop formation with probability 1 – p . R-loop formation could be initiated somewhere within the R-loop-prone sequence, but then the nascent RNA tail is likely to invade the entire R-loop-prone sequence (probably, even further upstream to the very start of transcription) as shown. The RNAP that created the R-loop could continue transcription in the ‘R-loop mode’, and then stall, either within, or at some distance downstream from the R-loop-prone sequence. At least some of the stalled RNAPs may remain bound to the DNA template (as shown), or could dissociate (not shown). In any case, R-loop formation blocks further rounds of transcription (the blockage is symbolized by the red crisscross). Addition of RNase H during transcription (all arrows that symbolize transitions within RNase H-related pathway are shown in green) leads to R-loop removal and, consequently, eliminates the blockage (blockage elimination is symbolized by the green path parallel to the crisscrossed path). The substrate DNA molecules from which R-loop was removed, then become available for further rounds of transcription, and would produce some number of normal full-sized transcripts, before an R-loop would form again. In addition, an RNAP stalled within an R-loop could resume transcription upon R-loop removal, producing a shorter transcript. That accounts for the pattern of transcription products obtained in the presence of RNase H (lane 4 in Figure 5 , the relevant part of it is placed in the present figure.).
    Figure Legend Snippet: Model for transcription blockage by R-loop formation in the vicinity of the promoter. The R-loop-prone (G-rich) DNA sequence is shown in turquoise, the rest of DNA is shown in gray, transcript from the R-loop-prone sequence is shown in dark blue, the rest of RNA is shown in black, a bent arrow indicates the transcription start site. RNA polymerase (RNAP) is shown as a gray circle. During transcription, an R-loop is formed with a certain probability p , while transcription proceeds without R-loop formation with probability 1 – p . R-loop formation could be initiated somewhere within the R-loop-prone sequence, but then the nascent RNA tail is likely to invade the entire R-loop-prone sequence (probably, even further upstream to the very start of transcription) as shown. The RNAP that created the R-loop could continue transcription in the ‘R-loop mode’, and then stall, either within, or at some distance downstream from the R-loop-prone sequence. At least some of the stalled RNAPs may remain bound to the DNA template (as shown), or could dissociate (not shown). In any case, R-loop formation blocks further rounds of transcription (the blockage is symbolized by the red crisscross). Addition of RNase H during transcription (all arrows that symbolize transitions within RNase H-related pathway are shown in green) leads to R-loop removal and, consequently, eliminates the blockage (blockage elimination is symbolized by the green path parallel to the crisscrossed path). The substrate DNA molecules from which R-loop was removed, then become available for further rounds of transcription, and would produce some number of normal full-sized transcripts, before an R-loop would form again. In addition, an RNAP stalled within an R-loop could resume transcription upon R-loop removal, producing a shorter transcript. That accounts for the pattern of transcription products obtained in the presence of RNase H (lane 4 in Figure 5 , the relevant part of it is placed in the present figure.).

    Techniques Used: Sequencing

    31) Product Images from "Strong transcription blockage mediated by R-loop formation within a G-rich homopurine–homopyrimidine sequence localized in the vicinity of the promoter"

    Article Title: Strong transcription blockage mediated by R-loop formation within a G-rich homopurine–homopyrimidine sequence localized in the vicinity of the promoter

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx403

    Effect of RNase H upon transcription. Substrates containing the G-rich sequence were used in these experiments. See the Results section for description of the experiment. ( A ) Gel image. ( B ) Quantitation of the results. All run-off signals are normalized to the signal for promoter–distal substrate transcribed without RNase H.
    Figure Legend Snippet: Effect of RNase H upon transcription. Substrates containing the G-rich sequence were used in these experiments. See the Results section for description of the experiment. ( A ) Gel image. ( B ) Quantitation of the results. All run-off signals are normalized to the signal for promoter–distal substrate transcribed without RNase H.

    Techniques Used: Sequencing, Quantitation Assay

    Model for transcription blockage by R-loop formation in the vicinity of the promoter. The R-loop-prone (G-rich) DNA sequence is shown in turquoise, the rest of DNA is shown in gray, transcript from the R-loop-prone sequence is shown in dark blue, the rest of RNA is shown in black, a bent arrow indicates the transcription start site. RNA polymerase (RNAP) is shown as a gray circle. During transcription, an R-loop is formed with a certain probability p , while transcription proceeds without R-loop formation with probability 1 – p . R-loop formation could be initiated somewhere within the R-loop-prone sequence, but then the nascent RNA tail is likely to invade the entire R-loop-prone sequence (probably, even further upstream to the very start of transcription) as shown. The RNAP that created the R-loop could continue transcription in the ‘R-loop mode’, and then stall, either within, or at some distance downstream from the R-loop-prone sequence. At least some of the stalled RNAPs may remain bound to the DNA template (as shown), or could dissociate (not shown). In any case, R-loop formation blocks further rounds of transcription (the blockage is symbolized by the red crisscross). Addition of RNase H during transcription (all arrows that symbolize transitions within RNase H-related pathway are shown in green) leads to R-loop removal and, consequently, eliminates the blockage (blockage elimination is symbolized by the green path parallel to the crisscrossed path). The substrate DNA molecules from which R-loop was removed, then become available for further rounds of transcription, and would produce some number of normal full-sized transcripts, before an R-loop would form again. In addition, an RNAP stalled within an R-loop could resume transcription upon R-loop removal, producing a shorter transcript. That accounts for the pattern of transcription products obtained in the presence of RNase H (lane 4 in Figure 5 , the relevant part of it is placed in the present figure.).
    Figure Legend Snippet: Model for transcription blockage by R-loop formation in the vicinity of the promoter. The R-loop-prone (G-rich) DNA sequence is shown in turquoise, the rest of DNA is shown in gray, transcript from the R-loop-prone sequence is shown in dark blue, the rest of RNA is shown in black, a bent arrow indicates the transcription start site. RNA polymerase (RNAP) is shown as a gray circle. During transcription, an R-loop is formed with a certain probability p , while transcription proceeds without R-loop formation with probability 1 – p . R-loop formation could be initiated somewhere within the R-loop-prone sequence, but then the nascent RNA tail is likely to invade the entire R-loop-prone sequence (probably, even further upstream to the very start of transcription) as shown. The RNAP that created the R-loop could continue transcription in the ‘R-loop mode’, and then stall, either within, or at some distance downstream from the R-loop-prone sequence. At least some of the stalled RNAPs may remain bound to the DNA template (as shown), or could dissociate (not shown). In any case, R-loop formation blocks further rounds of transcription (the blockage is symbolized by the red crisscross). Addition of RNase H during transcription (all arrows that symbolize transitions within RNase H-related pathway are shown in green) leads to R-loop removal and, consequently, eliminates the blockage (blockage elimination is symbolized by the green path parallel to the crisscrossed path). The substrate DNA molecules from which R-loop was removed, then become available for further rounds of transcription, and would produce some number of normal full-sized transcripts, before an R-loop would form again. In addition, an RNAP stalled within an R-loop could resume transcription upon R-loop removal, producing a shorter transcript. That accounts for the pattern of transcription products obtained in the presence of RNase H (lane 4 in Figure 5 , the relevant part of it is placed in the present figure.).

    Techniques Used: Sequencing

    32) Product Images from "Strong transcription blockage mediated by R-loop formation within a G-rich homopurine–homopyrimidine sequence localized in the vicinity of the promoter"

    Article Title: Strong transcription blockage mediated by R-loop formation within a G-rich homopurine–homopyrimidine sequence localized in the vicinity of the promoter

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx403

    Effect of RNase H upon transcription. Substrates containing the G-rich sequence were used in these experiments. See the Results section for description of the experiment. ( A ) Gel image. ( B ) Quantitation of the results. All run-off signals are normalized to the signal for promoter–distal substrate transcribed without RNase H.
    Figure Legend Snippet: Effect of RNase H upon transcription. Substrates containing the G-rich sequence were used in these experiments. See the Results section for description of the experiment. ( A ) Gel image. ( B ) Quantitation of the results. All run-off signals are normalized to the signal for promoter–distal substrate transcribed without RNase H.

    Techniques Used: Sequencing, Quantitation Assay

    Model for transcription blockage by R-loop formation in the vicinity of the promoter. The R-loop-prone (G-rich) DNA sequence is shown in turquoise, the rest of DNA is shown in gray, transcript from the R-loop-prone sequence is shown in dark blue, the rest of RNA is shown in black, a bent arrow indicates the transcription start site. RNA polymerase (RNAP) is shown as a gray circle. During transcription, an R-loop is formed with a certain probability p , while transcription proceeds without R-loop formation with probability 1 – p . R-loop formation could be initiated somewhere within the R-loop-prone sequence, but then the nascent RNA tail is likely to invade the entire R-loop-prone sequence (probably, even further upstream to the very start of transcription) as shown. The RNAP that created the R-loop could continue transcription in the ‘R-loop mode’, and then stall, either within, or at some distance downstream from the R-loop-prone sequence. At least some of the stalled RNAPs may remain bound to the DNA template (as shown), or could dissociate (not shown). In any case, R-loop formation blocks further rounds of transcription (the blockage is symbolized by the red crisscross). Addition of RNase H during transcription (all arrows that symbolize transitions within RNase H-related pathway are shown in green) leads to R-loop removal and, consequently, eliminates the blockage (blockage elimination is symbolized by the green path parallel to the crisscrossed path). The substrate DNA molecules from which R-loop was removed, then become available for further rounds of transcription, and would produce some number of normal full-sized transcripts, before an R-loop would form again. In addition, an RNAP stalled within an R-loop could resume transcription upon R-loop removal, producing a shorter transcript. That accounts for the pattern of transcription products obtained in the presence of RNase H (lane 4 in Figure 5 , the relevant part of it is placed in the present figure.).
    Figure Legend Snippet: Model for transcription blockage by R-loop formation in the vicinity of the promoter. The R-loop-prone (G-rich) DNA sequence is shown in turquoise, the rest of DNA is shown in gray, transcript from the R-loop-prone sequence is shown in dark blue, the rest of RNA is shown in black, a bent arrow indicates the transcription start site. RNA polymerase (RNAP) is shown as a gray circle. During transcription, an R-loop is formed with a certain probability p , while transcription proceeds without R-loop formation with probability 1 – p . R-loop formation could be initiated somewhere within the R-loop-prone sequence, but then the nascent RNA tail is likely to invade the entire R-loop-prone sequence (probably, even further upstream to the very start of transcription) as shown. The RNAP that created the R-loop could continue transcription in the ‘R-loop mode’, and then stall, either within, or at some distance downstream from the R-loop-prone sequence. At least some of the stalled RNAPs may remain bound to the DNA template (as shown), or could dissociate (not shown). In any case, R-loop formation blocks further rounds of transcription (the blockage is symbolized by the red crisscross). Addition of RNase H during transcription (all arrows that symbolize transitions within RNase H-related pathway are shown in green) leads to R-loop removal and, consequently, eliminates the blockage (blockage elimination is symbolized by the green path parallel to the crisscrossed path). The substrate DNA molecules from which R-loop was removed, then become available for further rounds of transcription, and would produce some number of normal full-sized transcripts, before an R-loop would form again. In addition, an RNAP stalled within an R-loop could resume transcription upon R-loop removal, producing a shorter transcript. That accounts for the pattern of transcription products obtained in the presence of RNase H (lane 4 in Figure 5 , the relevant part of it is placed in the present figure.).

    Techniques Used: Sequencing

    33) Product Images from "RNA from a simple-tandem repeat is required for sperm maturation and male fertility in Drosophila melanogaster"

    Article Title: RNA from a simple-tandem repeat is required for sperm maturation and male fertility in Drosophila melanogaster

    Journal: eLife

    doi: 10.7554/eLife.48940

    AAGAG RNA-FISH localizes RNA and not DNA. Confocal sections of cycle 14 nuclei treated with either ( a ) RNAseH or ( b ) RNAseIII after AAGAG RNA probe (magenta) hybridization. A higher laser intensity for the AAGAG probe channel was used in b to demonstrate abolishment of AAGAG signal.
    Figure Legend Snippet: AAGAG RNA-FISH localizes RNA and not DNA. Confocal sections of cycle 14 nuclei treated with either ( a ) RNAseH or ( b ) RNAseIII after AAGAG RNA probe (magenta) hybridization. A higher laser intensity for the AAGAG probe channel was used in b to demonstrate abolishment of AAGAG signal.

    Techniques Used: Fluorescence In Situ Hybridization, Hybridization

    AAGAG RNA foci contain single-stranded RNA and are not associated with R-loops. Confocal sections of embryonic nuclei in cycle 14 (with exception of left panel in ‘b’), nuclear periphery outlined in dotted circles. ( a ) No RNase control. ( b ) Treated with RNaseIII (left nucleus is cycle 12) ( c ) RNaseH ( d ) RNase1 and ( e ) RNaseA.
    Figure Legend Snippet: AAGAG RNA foci contain single-stranded RNA and are not associated with R-loops. Confocal sections of embryonic nuclei in cycle 14 (with exception of left panel in ‘b’), nuclear periphery outlined in dotted circles. ( a ) No RNase control. ( b ) Treated with RNaseIII (left nucleus is cycle 12) ( c ) RNaseH ( d ) RNase1 and ( e ) RNaseA.

    Techniques Used:

    34) Product Images from "An mRNA-binding channel in the ES6S region of the translation 48S-PIC promotes RNA unwinding and scanning"

    Article Title: An mRNA-binding channel in the ES6S region of the translation 48S-PIC promotes RNA unwinding and scanning

    Journal: eLife

    doi: 10.7554/eLife.48246

    Mapping and modeling of the oligo 4 bound to 48S-PIC. ( a ) Mapping oligo 4–18S pairing. RNAse H assay showing the cleavage fragments that are generated upon binding of oligo 4 and 5.4 to 40S subunits. It was determined that the last 6 nt of the 3′ end of VIC-oligo4 remained unpaired, but we could not determine whether the last two 5′ nt of oligo 4 were paired or not (lower panel). The target sequences for oligo 4 and 5.4 in the ES6S D and ES6S B helices, respectively, are marked in red (right panel). Note that in this human model of 40S, ES6S A is in the ‘outward’ conformation. ( b ) Model of TR–oligo 4 bound to PIC. The fluorophore is located between the ES6S A and ES6S D helices; note that in this model, ES6S A is in the ‘inward’ conformation. ( c ) Molecular structures of the IC, FITC and TR fluorophores.
    Figure Legend Snippet: Mapping and modeling of the oligo 4 bound to 48S-PIC. ( a ) Mapping oligo 4–18S pairing. RNAse H assay showing the cleavage fragments that are generated upon binding of oligo 4 and 5.4 to 40S subunits. It was determined that the last 6 nt of the 3′ end of VIC-oligo4 remained unpaired, but we could not determine whether the last two 5′ nt of oligo 4 were paired or not (lower panel). The target sequences for oligo 4 and 5.4 in the ES6S D and ES6S B helices, respectively, are marked in red (right panel). Note that in this human model of 40S, ES6S A is in the ‘outward’ conformation. ( b ) Model of TR–oligo 4 bound to PIC. The fluorophore is located between the ES6S A and ES6S D helices; note that in this model, ES6S A is in the ‘inward’ conformation. ( c ) Molecular structures of the IC, FITC and TR fluorophores.

    Techniques Used: Rnase H Assay, Generated, Binding Assay

    35) Product Images from "R-loops Associated with Triplet Repeat Expansions Promote Gene Silencing in Friedreich Ataxia and Fragile X Syndrome"

    Article Title: R-loops Associated with Triplet Repeat Expansions Promote Gene Silencing in Friedreich Ataxia and Fragile X Syndrome

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1004318

    R-loops are formed over (CGG) n expanded repeats of FMR1 gene. A. Diagram of FMR1 gene. Black boxes are exons, white box is 5′ UTR and lines are introns. Red triangle is (CGG) n expansion. qPCR amplicons are shown below the diagram. TSS is the transcriptional start site. Numbers indicate the distances to TSS in kilobases. B. RT-qPCR analysis of FMR1 mRNA in control and FXS cells, treated with 1 µM 5-azadC for 7 days, normalized to GAPDH. C. DIP analysis on endogenous FMR1 gene in control and FXS cells, treated with 1 µM 5-azadC for 7 days. Values are relative to ex1 region in control untreated cells. D. FMR1 R-loops are sensitive to RNase H digestion, following the treatment with 25 U of RNase H for 6 hours at 37°C prior to immuno-precipitation. Values are relative to in15 region in control untreated cells. E. R-loop kinetics on exon 1 of FMR1 gene in control and FXS cells during the process of transcriptional re-activation with 1 µM 5-azadC (7 days) followed by 5-azadC wash out with drug-free media (28 days). Values are relative to ex1 region in control untreated cells on day 7. F. RT-qPCR analysis of FMR1 mRNA in control and FXS cells, treated with 1 µM 5-azadC (7 days) followed by 5-azadC wash out with drug-free media (28 days). The level of FMR1 mRNA in control cells is taken as 1. Bars in B–D are average values +/− SEM (n > 3).
    Figure Legend Snippet: R-loops are formed over (CGG) n expanded repeats of FMR1 gene. A. Diagram of FMR1 gene. Black boxes are exons, white box is 5′ UTR and lines are introns. Red triangle is (CGG) n expansion. qPCR amplicons are shown below the diagram. TSS is the transcriptional start site. Numbers indicate the distances to TSS in kilobases. B. RT-qPCR analysis of FMR1 mRNA in control and FXS cells, treated with 1 µM 5-azadC for 7 days, normalized to GAPDH. C. DIP analysis on endogenous FMR1 gene in control and FXS cells, treated with 1 µM 5-azadC for 7 days. Values are relative to ex1 region in control untreated cells. D. FMR1 R-loops are sensitive to RNase H digestion, following the treatment with 25 U of RNase H for 6 hours at 37°C prior to immuno-precipitation. Values are relative to in15 region in control untreated cells. E. R-loop kinetics on exon 1 of FMR1 gene in control and FXS cells during the process of transcriptional re-activation with 1 µM 5-azadC (7 days) followed by 5-azadC wash out with drug-free media (28 days). Values are relative to ex1 region in control untreated cells on day 7. F. RT-qPCR analysis of FMR1 mRNA in control and FXS cells, treated with 1 µM 5-azadC (7 days) followed by 5-azadC wash out with drug-free media (28 days). The level of FMR1 mRNA in control cells is taken as 1. Bars in B–D are average values +/− SEM (n > 3).

    Techniques Used: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Immunoprecipitation, Activation Assay

    R-loops are formed over expanded repeats of FXN gene in FRDA cells. A. Diagram of FXN gene. Black boxes are exons, white boxes are 5′ and 3′UTRs, lines are introns, red triangle is (GAA) n expansion. TSS2 is the major transcriptional start site in lymphoblastoid cells. qPCR amplicons are shown below the diagram. Numbers indicate the distances to TSS2 in kilobases. B. Cell lines used in the study. The repeat sizes are indicated. C. RT-qPCR analysis of γ-actin, β-actin, GAPDH and FXN mRNAs in control (GM15851) and FRDA (GM15850) cells. Values are normalised to 5S rRNA and relative to control cells. D. RNA Pol II ChIP in control (GM15851) and FRDA (GM15850) cells. E. RT-qPCR analysis of FXN nascent RNA in control (GM15851) and FRDA (GM15850) cells, normalised to 5S rRNA and relative to ex1 RNA in control cells. F. DIP on endogenous FXN gene in control (GM15851) and FRDA (GM15851) cells. γ-actin is positive control. G. R-loops are sensitive to RNase H digestion. DIP samples were treated with 25 U of recombinant E.coli RNase H (NEB, M0297S) for 6 hours at 37°C. γ-actin is positive control. Bars in C–G are average values +/− SEM (n > 3).
    Figure Legend Snippet: R-loops are formed over expanded repeats of FXN gene in FRDA cells. A. Diagram of FXN gene. Black boxes are exons, white boxes are 5′ and 3′UTRs, lines are introns, red triangle is (GAA) n expansion. TSS2 is the major transcriptional start site in lymphoblastoid cells. qPCR amplicons are shown below the diagram. Numbers indicate the distances to TSS2 in kilobases. B. Cell lines used in the study. The repeat sizes are indicated. C. RT-qPCR analysis of γ-actin, β-actin, GAPDH and FXN mRNAs in control (GM15851) and FRDA (GM15850) cells. Values are normalised to 5S rRNA and relative to control cells. D. RNA Pol II ChIP in control (GM15851) and FRDA (GM15850) cells. E. RT-qPCR analysis of FXN nascent RNA in control (GM15851) and FRDA (GM15850) cells, normalised to 5S rRNA and relative to ex1 RNA in control cells. F. DIP on endogenous FXN gene in control (GM15851) and FRDA (GM15851) cells. γ-actin is positive control. G. R-loops are sensitive to RNase H digestion. DIP samples were treated with 25 U of recombinant E.coli RNase H (NEB, M0297S) for 6 hours at 37°C. γ-actin is positive control. Bars in C–G are average values +/− SEM (n > 3).

    Techniques Used: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Chromatin Immunoprecipitation, Positive Control, Recombinant

    36) Product Images from "RNase H eliminates R‐loops that disrupt DNA replication but is nonessential for efficient DSB repair"

    Article Title: RNase H eliminates R‐loops that disrupt DNA replication but is nonessential for efficient DSB repair

    Journal: EMBO Reports

    doi: 10.15252/embr.201745335

    RNase H‐deficient cells are weakly sensitive to camptothecin Plating assays indicate  rnh1 / 201∆  cells are weakly sensitive to CPT. Fivefold serial dilutions of cells were incubated on plates containing the indicated concentrations of CPT. Plates were photographed after 3‐day incubation at 32°C. Note  rad50∆  and  rnh1 / 201∆  cells form smaller colonies, indicating increased cell death. In transient exposure assays,  rnh1 / 201∆  cells are only weakly sensitive to CPT. Cells were exposed to 20 μM of CPT for 0–4 h. Bars represent standard deviation of three independent biological experiments. Plating assays indicate  rnh1 / 201∆  cells are moderately sensitive to HU. In transient exposure assays,  rnh1 / 201∆  cells are moderately sensitive to HU. Cells were treated with the indicated doses of HU for 6 h. Bars represent standard deviation of three independent biological experiments.
    Figure Legend Snippet: RNase H‐deficient cells are weakly sensitive to camptothecin Plating assays indicate rnh1 / 201∆ cells are weakly sensitive to CPT. Fivefold serial dilutions of cells were incubated on plates containing the indicated concentrations of CPT. Plates were photographed after 3‐day incubation at 32°C. Note rad50∆ and rnh1 / 201∆ cells form smaller colonies, indicating increased cell death. In transient exposure assays, rnh1 / 201∆ cells are only weakly sensitive to CPT. Cells were exposed to 20 μM of CPT for 0–4 h. Bars represent standard deviation of three independent biological experiments. Plating assays indicate rnh1 / 201∆ cells are moderately sensitive to HU. In transient exposure assays, rnh1 / 201∆ cells are moderately sensitive to HU. Cells were treated with the indicated doses of HU for 6 h. Bars represent standard deviation of three independent biological experiments.

    Techniques Used: Cycling Probe Technology, Incubation, Standard Deviation

    RNA–DNA hybrids are not enriched at the reparable mat1 DSB Diagram of the mat2,3∆ broken replication fork showing the location of PCR products used for ChIP and DRIP assays. Rad52 is enriched at the mat1 DSB site and the sister chromatid region used for HDR. ChIP assay was performed with Rad52‐5FLAG expressed from the endogenous locus in a mat2,3∆ strain. Relative enrichment was calculated as the percentage of ChIP/input and presented as the mean of three technical replicates. The results were replicated in three independent experiments. RNA–DNA hybrids are enriched at the tRNATyr gene, but not at the reparable DSB at the mat1 locus in rnh1 / 201∆ cells. DRIP assay was performed with S9.6 antibody using the indicated strains with or without RNase H treatment. Relative enrichment was calculated as ChIP/input. Relative enrichment was calculated as the percentage of ChIP/input and presented as the mean of three technical replicates. The results were replicated in three independent experiments.
    Figure Legend Snippet: RNA–DNA hybrids are not enriched at the reparable mat1 DSB Diagram of the mat2,3∆ broken replication fork showing the location of PCR products used for ChIP and DRIP assays. Rad52 is enriched at the mat1 DSB site and the sister chromatid region used for HDR. ChIP assay was performed with Rad52‐5FLAG expressed from the endogenous locus in a mat2,3∆ strain. Relative enrichment was calculated as the percentage of ChIP/input and presented as the mean of three technical replicates. The results were replicated in three independent experiments. RNA–DNA hybrids are enriched at the tRNATyr gene, but not at the reparable DSB at the mat1 locus in rnh1 / 201∆ cells. DRIP assay was performed with S9.6 antibody using the indicated strains with or without RNase H treatment. Relative enrichment was calculated as ChIP/input. Relative enrichment was calculated as the percentage of ChIP/input and presented as the mean of three technical replicates. The results were replicated in three independent experiments.

    Techniques Used: Polymerase Chain Reaction, Chromatin Immunoprecipitation

    HDR‐mediated reset of collapsed replication forks is essential in RNase H‐deficient cells Tetrad analysis showing that Rad50 is essential in  rnh1 / 201∆  background. Tetrad analysis showing that Ctp1 is essential in  rnh1 / 201∆  background. Tetrad analysis showing that Mus81 is essential in  rnh1 / 201∆  background.
    Figure Legend Snippet: HDR‐mediated reset of collapsed replication forks is essential in RNase H‐deficient cells Tetrad analysis showing that Rad50 is essential in rnh1 / 201∆ background. Tetrad analysis showing that Ctp1 is essential in rnh1 / 201∆ background. Tetrad analysis showing that Mus81 is essential in rnh1 / 201∆ background.

    Techniques Used:

    RNase H‐deficient cells are insensitive to ionizing radiation RNase H is not required for IR survival. Cells exposed to IR from a cesium‐137 source were plated with fivefold serial dilutions. Plates were photographed after 3‐day incubation at 32°C. Quantitative analysis confirms that rnh1 / 201∆ cells are insensitive to IR. Bars represent standard deviation of three independent biological experiments.
    Figure Legend Snippet: RNase H‐deficient cells are insensitive to ionizing radiation RNase H is not required for IR survival. Cells exposed to IR from a cesium‐137 source were plated with fivefold serial dilutions. Plates were photographed after 3‐day incubation at 32°C. Quantitative analysis confirms that rnh1 / 201∆ cells are insensitive to IR. Bars represent standard deviation of three independent biological experiments.

    Techniques Used: Incubation, Standard Deviation

    DSB repair at the mat1 broken replication fork occurs efficiently in the absence of RNase H Mating type switching system in fission yeast. See text for details. Lower panel shows repair mechanism in mat2,3∆ donorless strain. Proficient mating type switching in h 90 rnh1 / 201∆ cells. Colonies of the indicated genotypes on SSA plates were exposed to iodine vapor to assess mating/sporulation efficiency. Controls include wild‐type h 90 , non‐switchable h − , and switching‐defective swi3∆ h 90 . Rad50 is required SCR repair of the DSB at the mat1 locus in mat2,3∆ cells. The rad50∆ mat2,3∆ cells display very poor growth compared to single mutants or wild type. Products of a tetrad dissection were photographed on successive days. RNase H is not required for SCR repair of the DSB at the mat1 locus in mat2,3∆ cells. The rnh1 / 201∆ mat2,3∆ cells display no growth defect relative to rnh1∆ rnh201∆ .
    Figure Legend Snippet: DSB repair at the mat1 broken replication fork occurs efficiently in the absence of RNase H Mating type switching system in fission yeast. See text for details. Lower panel shows repair mechanism in mat2,3∆ donorless strain. Proficient mating type switching in h 90 rnh1 / 201∆ cells. Colonies of the indicated genotypes on SSA plates were exposed to iodine vapor to assess mating/sporulation efficiency. Controls include wild‐type h 90 , non‐switchable h − , and switching‐defective swi3∆ h 90 . Rad50 is required SCR repair of the DSB at the mat1 locus in mat2,3∆ cells. The rad50∆ mat2,3∆ cells display very poor growth compared to single mutants or wild type. Products of a tetrad dissection were photographed on successive days. RNase H is not required for SCR repair of the DSB at the mat1 locus in mat2,3∆ cells. The rnh1 / 201∆ mat2,3∆ cells display no growth defect relative to rnh1∆ rnh201∆ .

    Techniques Used: Dissection

    37) Product Images from "RNA:DNA hybrids are a novel molecular pattern sensed by TLR9"

    Article Title: RNA:DNA hybrids are a novel molecular pattern sensed by TLR9

    Journal: The EMBO Journal

    doi: 10.1002/embj.201386117

    Viral RNA:DNA hybrids accumulate in the cytoplasm and endosomes of MMLV infected cells. A, B Viral RNA:DNA hybrids are present in the cytoplasm of MMLV infected cells. (A) MMLV-specific PCR detects RNA:DNA hybrids immunoprecipitated from cytoplasmic nucleic acids. As PCR detects both MMLV DNA and RNA:DNA hybrids, specificity of the S9.6 pull down for hybrids is confirmed by the absence of PCR product in control samples processed with either S9.6 antibody omitted or when cytoplasmic nucleic acids were pretreated with RNase H. Labels: H 2 O, PCR without template added; Input, cytoplasmic nucleic acids prior to immunopreciptation (equal in proportion to post-pull down template); Uninfected, cytoplasmic nucleic acids from uninfected B3T3 cells. B Quantification of MMLV RNA:DNA hybrids immunoprecipitated by S9.6 by qPCR using two independent MMLV primer sets. S9.6 immunoprecipitate contains 4.1 ± 1.1% of input molecules. RNase H treatment reduces the amount of MMLV-DNA pulled down by S9.6 by > 90%. Data shown are the mean of three independent IP/qPCR experiments ± s.d. * P = 0.0366 (MMLV-1), * P = 0.231 (MMLV-2). C Validation of endosomal fractionation: the early endosome marker Rab5 is enriched in endosomal preparations. Western blotting of cytoplasmic and endosomal fractions shows the presence of the endosomal marker Rab5 in both endosomal and cytoplasmic fractions, whereas GAPDH is only present in cytoplasmic fractions. Densitometry measurements show that relative Rab5 enrichment is > 22-fold relative to GAPDH. D Viral RNA:DNA hybrids are present in endosomal fractions of MMLV-infected cells. MMLV DNA was detected by PCR after S9.6 pull-down of hybrids from endosomal nucleic acids, but not in beads only or RNase H treated controls.
    Figure Legend Snippet: Viral RNA:DNA hybrids accumulate in the cytoplasm and endosomes of MMLV infected cells. A, B Viral RNA:DNA hybrids are present in the cytoplasm of MMLV infected cells. (A) MMLV-specific PCR detects RNA:DNA hybrids immunoprecipitated from cytoplasmic nucleic acids. As PCR detects both MMLV DNA and RNA:DNA hybrids, specificity of the S9.6 pull down for hybrids is confirmed by the absence of PCR product in control samples processed with either S9.6 antibody omitted or when cytoplasmic nucleic acids were pretreated with RNase H. Labels: H 2 O, PCR without template added; Input, cytoplasmic nucleic acids prior to immunopreciptation (equal in proportion to post-pull down template); Uninfected, cytoplasmic nucleic acids from uninfected B3T3 cells. B Quantification of MMLV RNA:DNA hybrids immunoprecipitated by S9.6 by qPCR using two independent MMLV primer sets. S9.6 immunoprecipitate contains 4.1 ± 1.1% of input molecules. RNase H treatment reduces the amount of MMLV-DNA pulled down by S9.6 by > 90%. Data shown are the mean of three independent IP/qPCR experiments ± s.d. * P = 0.0366 (MMLV-1), * P = 0.231 (MMLV-2). C Validation of endosomal fractionation: the early endosome marker Rab5 is enriched in endosomal preparations. Western blotting of cytoplasmic and endosomal fractions shows the presence of the endosomal marker Rab5 in both endosomal and cytoplasmic fractions, whereas GAPDH is only present in cytoplasmic fractions. Densitometry measurements show that relative Rab5 enrichment is > 22-fold relative to GAPDH. D Viral RNA:DNA hybrids are present in endosomal fractions of MMLV-infected cells. MMLV DNA was detected by PCR after S9.6 pull-down of hybrids from endosomal nucleic acids, but not in beads only or RNase H treated controls.

    Techniques Used: Infection, Polymerase Chain Reaction, Immunoprecipitation, Real-time Polymerase Chain Reaction, Fractionation, Marker, Western Blot

    38) Product Images from "Defining the location of promoter-associated R-loops at near-nucleotide resolution using bisDRIP-seq"

    Article Title: Defining the location of promoter-associated R-loops at near-nucleotide resolution using bisDRIP-seq

    Journal: eLife

    doi: 10.7554/eLife.28306

    bisDRIP-seq scores are sensitive to RNase H. ( A ) RNA-DNA hybrid formation is necessary for enrichment of bisDRIP-seq signal at sites of R-loop formation. RNA-DNA hybrids are integral components of R-loops. RNA-DNA hybrids are known to bind the S9.6 antibody and to be degraded by RNase H. R-loop signal should therefore be lost in the absence of S9.6 immunoprecipitation or if the sample is treated with RNase H. In each of these plots, the template strand refers to the strand used for RPPH1 transcription, rather than the template strand used for PAPR2 transcription. Also, in each plot the bisDRIP-seq score on the template strand and non-template strand were plotted below the x-axis (orange) or above the x-axis (blue), respectively. In the top plot, bisDRIP-seq scores were mapped to the genomic region surrounding RPPH1 from ‘input’ samples (mean bisDRIP-seq score from n = 2 samples). These input samples received the entire bisDRIP-seq treatment except for the S9.6 immunoprecipitation enrichment steps. In the middle plot, bisDRIP-seq scores were mapped to the genomic region surrounding RPPH1 from a control-treated bisDRIP-seq experiment that was not treated with RNase H (n = 1 sample). In the bottom plot, bisDRIP-seq scores were mapped to the genomic region surrounding RPPH1 from an RNase H-treated bisDRIP-seq experiment (n = 1 sample). The bisDRIP-seq sample that was not treated with RNase H has high bisDRIP-seq score at the RPPH1 locus. On the other hand, there is a large decrease in bisDRIP-seq signal in the sample treated with RNase H. This suggests that bisDRIP-seq signal in the RPPH1 locus depends on the presence of an RNA-DNA hybrid.
    Figure Legend Snippet: bisDRIP-seq scores are sensitive to RNase H. ( A ) RNA-DNA hybrid formation is necessary for enrichment of bisDRIP-seq signal at sites of R-loop formation. RNA-DNA hybrids are integral components of R-loops. RNA-DNA hybrids are known to bind the S9.6 antibody and to be degraded by RNase H. R-loop signal should therefore be lost in the absence of S9.6 immunoprecipitation or if the sample is treated with RNase H. In each of these plots, the template strand refers to the strand used for RPPH1 transcription, rather than the template strand used for PAPR2 transcription. Also, in each plot the bisDRIP-seq score on the template strand and non-template strand were plotted below the x-axis (orange) or above the x-axis (blue), respectively. In the top plot, bisDRIP-seq scores were mapped to the genomic region surrounding RPPH1 from ‘input’ samples (mean bisDRIP-seq score from n = 2 samples). These input samples received the entire bisDRIP-seq treatment except for the S9.6 immunoprecipitation enrichment steps. In the middle plot, bisDRIP-seq scores were mapped to the genomic region surrounding RPPH1 from a control-treated bisDRIP-seq experiment that was not treated with RNase H (n = 1 sample). In the bottom plot, bisDRIP-seq scores were mapped to the genomic region surrounding RPPH1 from an RNase H-treated bisDRIP-seq experiment (n = 1 sample). The bisDRIP-seq sample that was not treated with RNase H has high bisDRIP-seq score at the RPPH1 locus. On the other hand, there is a large decrease in bisDRIP-seq signal in the sample treated with RNase H. This suggests that bisDRIP-seq signal in the RPPH1 locus depends on the presence of an RNA-DNA hybrid.

    Techniques Used: Immunoprecipitation

    The strand asymmetry of bisDRIP-seq scores 3' of the transcription start site is sensitive to RNase H. ( A,B ) The strand asymmetry in bisDRIP-seq scores 3' of transcription start sites is not observed after RNase H treatment. RNA-DNA hybrids are core components of R-loops. If the strand asymmetry observed in metaplots of promoter regions is due to R-loops, than it should be selectively sensitive to RNase H. We therefore generated metaplots of bisDRIP-seq scores from two samples treated identically except that ( A ) received a vehicle treatment, while ( B ) was treated with RNase H. Metaplots were created by summing the bisDRIP-seq scores across active promoter regions (n = 15644) at each nucleotide position relative to the transcription start site. bisDRIP-seq scores were calculated separately for the nucleotide position on the non-template strand (blue) and template strand (orange). In the vehicle-treated sample, non-template bisDRIP-seq scores are higher than template bisDRIP-seq scores immediately 3' of the transcription start site. This replicates the result from Figure 3E . On the other hand, there is a complete loss of bisDRIP-seq score strand asymmetry after RNase H treatment in ( B ). This confirms that the asymmetry observed in Figure 3E is caused by R-loops.
    Figure Legend Snippet: The strand asymmetry of bisDRIP-seq scores 3' of the transcription start site is sensitive to RNase H. ( A,B ) The strand asymmetry in bisDRIP-seq scores 3' of transcription start sites is not observed after RNase H treatment. RNA-DNA hybrids are core components of R-loops. If the strand asymmetry observed in metaplots of promoter regions is due to R-loops, than it should be selectively sensitive to RNase H. We therefore generated metaplots of bisDRIP-seq scores from two samples treated identically except that ( A ) received a vehicle treatment, while ( B ) was treated with RNase H. Metaplots were created by summing the bisDRIP-seq scores across active promoter regions (n = 15644) at each nucleotide position relative to the transcription start site. bisDRIP-seq scores were calculated separately for the nucleotide position on the non-template strand (blue) and template strand (orange). In the vehicle-treated sample, non-template bisDRIP-seq scores are higher than template bisDRIP-seq scores immediately 3' of the transcription start site. This replicates the result from Figure 3E . On the other hand, there is a complete loss of bisDRIP-seq score strand asymmetry after RNase H treatment in ( B ). This confirms that the asymmetry observed in Figure 3E is caused by R-loops.

    Techniques Used: Generated

    R-loop signal in MALAT1 and NEAT1 is reduced by RNase H treatment. ( A,B ) bisDRIP-seq enrichment at ( A ) MALAT1 and ( B ) NEAT1 is lost after RNase H treatment and is not observed without RNA-DNA hybrid immunoprecipitation by S9.6 antibody. RNase H should selectively degrade the RNA component of R-loops. Thus, if bisDRIP-seq signal is caused by R-loops, RNase H treatment should reduce bisDRIP-seq signal. In the top panel, bisDRIP-seq scores from ‘input’ samples (mean of n = 2 samples) were mapped to each gene locus. bisDRIP-seq input samples were treated an identical manner to other bisDRIP-seq samples, except that no S9.6 antibody immunoprecipitation steps were performed. In the middle and lower plots, the bisDRIP-seq scores from two matched bisDRIP-seq experiments were mapped to each locus. The only difference between these experiments is that one sample was not treated with RNase H (middle plots, n = 1 sample), while the other sample was treated with RNase H (lower plots, n = 1 sample). At both loci, the sample that was not treated with RNase H replicated the bisDRIP-seq signal observed in standard bisDRIP-seq control experiments ( Figure 6A and B ). On the other hand, there is less bisDRIP-seq signal observed at each loci in both the input and RNase H-treated samples. These results suggest that RNA-DNA hybrids form in the MALAT1 and NEAT1 loci and that these RNA-DNA hybrids cause the high bisDRIP-seq scores observed at these loci. The location of the transcription start site is demarcated by ‘TSS’ and a dashed vertical line.
    Figure Legend Snippet: R-loop signal in MALAT1 and NEAT1 is reduced by RNase H treatment. ( A,B ) bisDRIP-seq enrichment at ( A ) MALAT1 and ( B ) NEAT1 is lost after RNase H treatment and is not observed without RNA-DNA hybrid immunoprecipitation by S9.6 antibody. RNase H should selectively degrade the RNA component of R-loops. Thus, if bisDRIP-seq signal is caused by R-loops, RNase H treatment should reduce bisDRIP-seq signal. In the top panel, bisDRIP-seq scores from ‘input’ samples (mean of n = 2 samples) were mapped to each gene locus. bisDRIP-seq input samples were treated an identical manner to other bisDRIP-seq samples, except that no S9.6 antibody immunoprecipitation steps were performed. In the middle and lower plots, the bisDRIP-seq scores from two matched bisDRIP-seq experiments were mapped to each locus. The only difference between these experiments is that one sample was not treated with RNase H (middle plots, n = 1 sample), while the other sample was treated with RNase H (lower plots, n = 1 sample). At both loci, the sample that was not treated with RNase H replicated the bisDRIP-seq signal observed in standard bisDRIP-seq control experiments ( Figure 6A and B ). On the other hand, there is less bisDRIP-seq signal observed at each loci in both the input and RNase H-treated samples. These results suggest that RNA-DNA hybrids form in the MALAT1 and NEAT1 loci and that these RNA-DNA hybrids cause the high bisDRIP-seq scores observed at these loci. The location of the transcription start site is demarcated by ‘TSS’ and a dashed vertical line.

    Techniques Used: Immunoprecipitation

    Histone genes are enriched in R-loops. ( A,B ) RNA polymerase II binding and RNA levels in the nucleus do not appear to explain the association between histone genes and R-loop formation. R-loop formation was calculated for each promoter region by taking the sum of bisDRIP-seq score strand asymmetry and the triptolide-sensitivity the non-template bisDRIP-seq scores. R-loop formation was then plotted against ( A ) nuclear RNA levels or ( B ) POLR2A (RNA polymerase II) chromatin immunoprecipitation (ChIP) signal for each promoter region. Promoter regions were plotted separately for non-histone genes (blue) and replication-dependent histone genes (orange). A linear regression for all genes was also plotted (blue). Both nuclear RNA levels and RNA polymerase II ChIP signal were weakly correlated with R-loop formation. Nevertheless, replication-dependent histone genes typically had higher R-loop formation scores than genes with similar promoter activity. This suggests that the nuclear RNA level of histones mRNAs in do not explain the high R-loop signal of these genes. It also suggests that the level of RNA polymerase II bound to histone genes does not explain the high R-loop signal observed at these genes. The bisDRIP-seq values in both plots were derived from the mean bisDRIP-seq scores of n = 13 control-treated samples and mean bisDRIP-seq scores of n = 2 triptolide-treated samples. In ( A ), we examined n = 1813 non-histone genes and n = 2 histone genes. In ( B ), we examined n = 2064 non-histone genes and n = 13 histone genes. TPKM refers to transcripts per kilobase million. The shaded areas around linear regression models represent 95% confidence intervals. ( C ) RNase H sensitivity of the bisDRIP-seq signal in histone genes. Metaplots were generated of histone gene non-template bisDRIP-seq score from three different bisDRIP-seq experiments. In the top plot, bisDRIP-seq scores were derived from ‘input’ samples (mean bisDRIP-seq score from n = 2 samples). These input samples received the entire bisDRIP-seq treatment except for the S9.6 immunoprecipitation enrichment steps. In the middle plot, bisDRIP-seq scores were derived from a bisDRIP-seq experiment that did not include an RNase H treatment (n = 1 sample). In the bottom plot, bisDRIP-seq scores were derived from an RNase H-treated bisDRIP-seq experiment (n = 1 sample). The bisDRIP-seq sample that was not treated with RNase H replicated the high non-template bisDRIP-seq scores observed in other bisDRIP-seq experiments at histone loci. On the other hand, almost no bisDRIP-seq signal was observed in either the input samples or in the bisDRIP-seq sample treated with RNase H. This suggests that the bisDRIP-seq signal observed at these gene loci is caused by R-loops. Transcription start sites are indicated by both ‘TSS’ and by dashed vertical lines. ( D,E ) bisDRIP-seq enrichment at ( D ) HIST1H2BG and ( E ) HIST1H1E is due to the presence of R-loops. In the middle and top plots, we compare the bisDRIP-seq from an RNase H treated sample (n = 1) and a bisDRIP-seq sample that was not treated with RNase H (n = 1), respectively. In both the middle and top plot, bisDRIP-seq scores from each experiment were mapped to the genomic region surrounding ( D ) HIST1H2BG and ( E ) HIST1H1E . At both loci, the bisDRIP-seq sample that was not treated with RNase H had similar bisDRIP-seq signal to our other bisDRIP-seq control experiments ( Figure 5D and E ). On the other hand, there is a clear reduction in bisDRIP-seq scores in the RNase H-treated sample. This decrease in signal after RNase H treatment suggests that RNA-DNA hybrids do form at these loci. In the bottom panel, the percentage of each nucleotide is plotted in 51 bp windows. There is no obvious relationship between nucleotide composition and bisDRIP-seq scores throughout these two genes.
    Figure Legend Snippet: Histone genes are enriched in R-loops. ( A,B ) RNA polymerase II binding and RNA levels in the nucleus do not appear to explain the association between histone genes and R-loop formation. R-loop formation was calculated for each promoter region by taking the sum of bisDRIP-seq score strand asymmetry and the triptolide-sensitivity the non-template bisDRIP-seq scores. R-loop formation was then plotted against ( A ) nuclear RNA levels or ( B ) POLR2A (RNA polymerase II) chromatin immunoprecipitation (ChIP) signal for each promoter region. Promoter regions were plotted separately for non-histone genes (blue) and replication-dependent histone genes (orange). A linear regression for all genes was also plotted (blue). Both nuclear RNA levels and RNA polymerase II ChIP signal were weakly correlated with R-loop formation. Nevertheless, replication-dependent histone genes typically had higher R-loop formation scores than genes with similar promoter activity. This suggests that the nuclear RNA level of histones mRNAs in do not explain the high R-loop signal of these genes. It also suggests that the level of RNA polymerase II bound to histone genes does not explain the high R-loop signal observed at these genes. The bisDRIP-seq values in both plots were derived from the mean bisDRIP-seq scores of n = 13 control-treated samples and mean bisDRIP-seq scores of n = 2 triptolide-treated samples. In ( A ), we examined n = 1813 non-histone genes and n = 2 histone genes. In ( B ), we examined n = 2064 non-histone genes and n = 13 histone genes. TPKM refers to transcripts per kilobase million. The shaded areas around linear regression models represent 95% confidence intervals. ( C ) RNase H sensitivity of the bisDRIP-seq signal in histone genes. Metaplots were generated of histone gene non-template bisDRIP-seq score from three different bisDRIP-seq experiments. In the top plot, bisDRIP-seq scores were derived from ‘input’ samples (mean bisDRIP-seq score from n = 2 samples). These input samples received the entire bisDRIP-seq treatment except for the S9.6 immunoprecipitation enrichment steps. In the middle plot, bisDRIP-seq scores were derived from a bisDRIP-seq experiment that did not include an RNase H treatment (n = 1 sample). In the bottom plot, bisDRIP-seq scores were derived from an RNase H-treated bisDRIP-seq experiment (n = 1 sample). The bisDRIP-seq sample that was not treated with RNase H replicated the high non-template bisDRIP-seq scores observed in other bisDRIP-seq experiments at histone loci. On the other hand, almost no bisDRIP-seq signal was observed in either the input samples or in the bisDRIP-seq sample treated with RNase H. This suggests that the bisDRIP-seq signal observed at these gene loci is caused by R-loops. Transcription start sites are indicated by both ‘TSS’ and by dashed vertical lines. ( D,E ) bisDRIP-seq enrichment at ( D ) HIST1H2BG and ( E ) HIST1H1E is due to the presence of R-loops. In the middle and top plots, we compare the bisDRIP-seq from an RNase H treated sample (n = 1) and a bisDRIP-seq sample that was not treated with RNase H (n = 1), respectively. In both the middle and top plot, bisDRIP-seq scores from each experiment were mapped to the genomic region surrounding ( D ) HIST1H2BG and ( E ) HIST1H1E . At both loci, the bisDRIP-seq sample that was not treated with RNase H had similar bisDRIP-seq signal to our other bisDRIP-seq control experiments ( Figure 5D and E ). On the other hand, there is a clear reduction in bisDRIP-seq scores in the RNase H-treated sample. This decrease in signal after RNase H treatment suggests that RNA-DNA hybrids do form at these loci. In the bottom panel, the percentage of each nucleotide is plotted in 51 bp windows. There is no obvious relationship between nucleotide composition and bisDRIP-seq scores throughout these two genes.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Activity Assay, Derivative Assay, Generated, Immunoprecipitation

    39) Product Images from "Defining the location of promoter-associated R-loops at near-nucleotide resolution using bisDRIP-seq"

    Article Title: Defining the location of promoter-associated R-loops at near-nucleotide resolution using bisDRIP-seq

    Journal: eLife

    doi: 10.7554/eLife.28306

    bisDRIP-seq scores are sensitive to RNase H. ( A ) RNA-DNA hybrid formation is necessary for enrichment of bisDRIP-seq signal at sites of R-loop formation. RNA-DNA hybrids are integral components of R-loops. RNA-DNA hybrids are known to bind the S9.6 antibody and to be degraded by RNase H. R-loop signal should therefore be lost in the absence of S9.6 immunoprecipitation or if the sample is treated with RNase H. In each of these plots, the template strand refers to the strand used for RPPH1 transcription, rather than the template strand used for PAPR2 transcription. Also, in each plot the bisDRIP-seq score on the template strand and non-template strand were plotted below the x-axis (orange) or above the x-axis (blue), respectively. In the top plot, bisDRIP-seq scores were mapped to the genomic region surrounding RPPH1 from ‘input’ samples (mean bisDRIP-seq score from n = 2 samples). These input samples received the entire bisDRIP-seq treatment except for the S9.6 immunoprecipitation enrichment steps. In the middle plot, bisDRIP-seq scores were mapped to the genomic region surrounding RPPH1 from a control-treated bisDRIP-seq experiment that was not treated with RNase H (n = 1 sample). In the bottom plot, bisDRIP-seq scores were mapped to the genomic region surrounding RPPH1 from an RNase H-treated bisDRIP-seq experiment (n = 1 sample). The bisDRIP-seq sample that was not treated with RNase H has high bisDRIP-seq score at the RPPH1 locus. On the other hand, there is a large decrease in bisDRIP-seq signal in the sample treated with RNase H. This suggests that bisDRIP-seq signal in the RPPH1 locus depends on the presence of an RNA-DNA hybrid.
    Figure Legend Snippet: bisDRIP-seq scores are sensitive to RNase H. ( A ) RNA-DNA hybrid formation is necessary for enrichment of bisDRIP-seq signal at sites of R-loop formation. RNA-DNA hybrids are integral components of R-loops. RNA-DNA hybrids are known to bind the S9.6 antibody and to be degraded by RNase H. R-loop signal should therefore be lost in the absence of S9.6 immunoprecipitation or if the sample is treated with RNase H. In each of these plots, the template strand refers to the strand used for RPPH1 transcription, rather than the template strand used for PAPR2 transcription. Also, in each plot the bisDRIP-seq score on the template strand and non-template strand were plotted below the x-axis (orange) or above the x-axis (blue), respectively. In the top plot, bisDRIP-seq scores were mapped to the genomic region surrounding RPPH1 from ‘input’ samples (mean bisDRIP-seq score from n = 2 samples). These input samples received the entire bisDRIP-seq treatment except for the S9.6 immunoprecipitation enrichment steps. In the middle plot, bisDRIP-seq scores were mapped to the genomic region surrounding RPPH1 from a control-treated bisDRIP-seq experiment that was not treated with RNase H (n = 1 sample). In the bottom plot, bisDRIP-seq scores were mapped to the genomic region surrounding RPPH1 from an RNase H-treated bisDRIP-seq experiment (n = 1 sample). The bisDRIP-seq sample that was not treated with RNase H has high bisDRIP-seq score at the RPPH1 locus. On the other hand, there is a large decrease in bisDRIP-seq signal in the sample treated with RNase H. This suggests that bisDRIP-seq signal in the RPPH1 locus depends on the presence of an RNA-DNA hybrid.

    Techniques Used: Immunoprecipitation

    The strand asymmetry of bisDRIP-seq scores 3' of the transcription start site is sensitive to RNase H. ( A,B ) The strand asymmetry in bisDRIP-seq scores 3' of transcription start sites is not observed after RNase H treatment. RNA-DNA hybrids are core components of R-loops. If the strand asymmetry observed in metaplots of promoter regions is due to R-loops, than it should be selectively sensitive to RNase H. We therefore generated metaplots of bisDRIP-seq scores from two samples treated identically except that ( A ) received a vehicle treatment, while ( B ) was treated with RNase H. Metaplots were created by summing the bisDRIP-seq scores across active promoter regions (n = 15644) at each nucleotide position relative to the transcription start site. bisDRIP-seq scores were calculated separately for the nucleotide position on the non-template strand (blue) and template strand (orange). In the vehicle-treated sample, non-template bisDRIP-seq scores are higher than template bisDRIP-seq scores immediately 3' of the transcription start site. This replicates the result from Figure 3E . On the other hand, there is a complete loss of bisDRIP-seq score strand asymmetry after RNase H treatment in ( B ). This confirms that the asymmetry observed in Figure 3E is caused by R-loops.
    Figure Legend Snippet: The strand asymmetry of bisDRIP-seq scores 3' of the transcription start site is sensitive to RNase H. ( A,B ) The strand asymmetry in bisDRIP-seq scores 3' of transcription start sites is not observed after RNase H treatment. RNA-DNA hybrids are core components of R-loops. If the strand asymmetry observed in metaplots of promoter regions is due to R-loops, than it should be selectively sensitive to RNase H. We therefore generated metaplots of bisDRIP-seq scores from two samples treated identically except that ( A ) received a vehicle treatment, while ( B ) was treated with RNase H. Metaplots were created by summing the bisDRIP-seq scores across active promoter regions (n = 15644) at each nucleotide position relative to the transcription start site. bisDRIP-seq scores were calculated separately for the nucleotide position on the non-template strand (blue) and template strand (orange). In the vehicle-treated sample, non-template bisDRIP-seq scores are higher than template bisDRIP-seq scores immediately 3' of the transcription start site. This replicates the result from Figure 3E . On the other hand, there is a complete loss of bisDRIP-seq score strand asymmetry after RNase H treatment in ( B ). This confirms that the asymmetry observed in Figure 3E is caused by R-loops.

    Techniques Used: Generated

    R-loop signal in MALAT1 and NEAT1 is reduced by RNase H treatment. ( A,B ) bisDRIP-seq enrichment at ( A ) MALAT1 and ( B ) NEAT1 is lost after RNase H treatment and is not observed without RNA-DNA hybrid immunoprecipitation by S9.6 antibody. RNase H should selectively degrade the RNA component of R-loops. Thus, if bisDRIP-seq signal is caused by R-loops, RNase H treatment should reduce bisDRIP-seq signal. In the top panel, bisDRIP-seq scores from ‘input’ samples (mean of n = 2 samples) were mapped to each gene locus. bisDRIP-seq input samples were treated an identical manner to other bisDRIP-seq samples, except that no S9.6 antibody immunoprecipitation steps were performed. In the middle and lower plots, the bisDRIP-seq scores from two matched bisDRIP-seq experiments were mapped to each locus. The only difference between these experiments is that one sample was not treated with RNase H (middle plots, n = 1 sample), while the other sample was treated with RNase H (lower plots, n = 1 sample). At both loci, the sample that was not treated with RNase H replicated the bisDRIP-seq signal observed in standard bisDRIP-seq control experiments ( Figure 6A and B ). On the other hand, there is less bisDRIP-seq signal observed at each loci in both the input and RNase H-treated samples. These results suggest that RNA-DNA hybrids form in the MALAT1 and NEAT1 loci and that these RNA-DNA hybrids cause the high bisDRIP-seq scores observed at these loci. The location of the transcription start site is demarcated by ‘TSS’ and a dashed vertical line.
    Figure Legend Snippet: R-loop signal in MALAT1 and NEAT1 is reduced by RNase H treatment. ( A,B ) bisDRIP-seq enrichment at ( A ) MALAT1 and ( B ) NEAT1 is lost after RNase H treatment and is not observed without RNA-DNA hybrid immunoprecipitation by S9.6 antibody. RNase H should selectively degrade the RNA component of R-loops. Thus, if bisDRIP-seq signal is caused by R-loops, RNase H treatment should reduce bisDRIP-seq signal. In the top panel, bisDRIP-seq scores from ‘input’ samples (mean of n = 2 samples) were mapped to each gene locus. bisDRIP-seq input samples were treated an identical manner to other bisDRIP-seq samples, except that no S9.6 antibody immunoprecipitation steps were performed. In the middle and lower plots, the bisDRIP-seq scores from two matched bisDRIP-seq experiments were mapped to each locus. The only difference between these experiments is that one sample was not treated with RNase H (middle plots, n = 1 sample), while the other sample was treated with RNase H (lower plots, n = 1 sample). At both loci, the sample that was not treated with RNase H replicated the bisDRIP-seq signal observed in standard bisDRIP-seq control experiments ( Figure 6A and B ). On the other hand, there is less bisDRIP-seq signal observed at each loci in both the input and RNase H-treated samples. These results suggest that RNA-DNA hybrids form in the MALAT1 and NEAT1 loci and that these RNA-DNA hybrids cause the high bisDRIP-seq scores observed at these loci. The location of the transcription start site is demarcated by ‘TSS’ and a dashed vertical line.

    Techniques Used: Immunoprecipitation

    Histone genes are enriched in R-loops. ( A,B ) RNA polymerase II binding and RNA levels in the nucleus do not appear to explain the association between histone genes and R-loop formation. R-loop formation was calculated for each promoter region by taking the sum of bisDRIP-seq score strand asymmetry and the triptolide-sensitivity the non-template bisDRIP-seq scores. R-loop formation was then plotted against ( A ) nuclear RNA levels or ( B ) POLR2A (RNA polymerase II) chromatin immunoprecipitation (ChIP) signal for each promoter region. Promoter regions were plotted separately for non-histone genes (blue) and replication-dependent histone genes (orange). A linear regression for all genes was also plotted (blue). Both nuclear RNA levels and RNA polymerase II ChIP signal were weakly correlated with R-loop formation. Nevertheless, replication-dependent histone genes typically had higher R-loop formation scores than genes with similar promoter activity. This suggests that the nuclear RNA level of histones mRNAs in do not explain the high R-loop signal of these genes. It also suggests that the level of RNA polymerase II bound to histone genes does not explain the high R-loop signal observed at these genes. The bisDRIP-seq values in both plots were derived from the mean bisDRIP-seq scores of n = 13 control-treated samples and mean bisDRIP-seq scores of n = 2 triptolide-treated samples. In ( A ), we examined n = 1813 non-histone genes and n = 2 histone genes. In ( B ), we examined n = 2064 non-histone genes and n = 13 histone genes. TPKM refers to transcripts per kilobase million. The shaded areas around linear regression models represent 95% confidence intervals. ( C ) RNase H sensitivity of the bisDRIP-seq signal in histone genes. Metaplots were generated of histone gene non-template bisDRIP-seq score from three different bisDRIP-seq experiments. In the top plot, bisDRIP-seq scores were derived from ‘input’ samples (mean bisDRIP-seq score from n = 2 samples). These input samples received the entire bisDRIP-seq treatment except for the S9.6 immunoprecipitation enrichment steps. In the middle plot, bisDRIP-seq scores were derived from a bisDRIP-seq experiment that did not include an RNase H treatment (n = 1 sample). In the bottom plot, bisDRIP-seq scores were derived from an RNase H-treated bisDRIP-seq experiment (n = 1 sample). The bisDRIP-seq sample that was not treated with RNase H replicated the high non-template bisDRIP-seq scores observed in other bisDRIP-seq experiments at histone loci. On the other hand, almost no bisDRIP-seq signal was observed in either the input samples or in the bisDRIP-seq sample treated with RNase H. This suggests that the bisDRIP-seq signal observed at these gene loci is caused by R-loops. Transcription start sites are indicated by both ‘TSS’ and by dashed vertical lines. ( D,E ) bisDRIP-seq enrichment at ( D ) HIST1H2BG and ( E ) HIST1H1E is due to the presence of R-loops. In the middle and top plots, we compare the bisDRIP-seq from an RNase H treated sample (n = 1) and a bisDRIP-seq sample that was not treated with RNase H (n = 1), respectively. In both the middle and top plot, bisDRIP-seq scores from each experiment were mapped to the genomic region surrounding ( D ) HIST1H2BG and ( E ) HIST1H1E . At both loci, the bisDRIP-seq sample that was not treated with RNase H had similar bisDRIP-seq signal to our other bisDRIP-seq control experiments ( Figure 5D and E ). On the other hand, there is a clear reduction in bisDRIP-seq scores in the RNase H-treated sample. This decrease in signal after RNase H treatment suggests that RNA-DNA hybrids do form at these loci. In the bottom panel, the percentage of each nucleotide is plotted in 51 bp windows. There is no obvious relationship between nucleotide composition and bisDRIP-seq scores throughout these two genes.
    Figure Legend Snippet: Histone genes are enriched in R-loops. ( A,B ) RNA polymerase II binding and RNA levels in the nucleus do not appear to explain the association between histone genes and R-loop formation. R-loop formation was calculated for each promoter region by taking the sum of bisDRIP-seq score strand asymmetry and the triptolide-sensitivity the non-template bisDRIP-seq scores. R-loop formation was then plotted against ( A ) nuclear RNA levels or ( B ) POLR2A (RNA polymerase II) chromatin immunoprecipitation (ChIP) signal for each promoter region. Promoter regions were plotted separately for non-histone genes (blue) and replication-dependent histone genes (orange). A linear regression for all genes was also plotted (blue). Both nuclear RNA levels and RNA polymerase II ChIP signal were weakly correlated with R-loop formation. Nevertheless, replication-dependent histone genes typically had higher R-loop formation scores than genes with similar promoter activity. This suggests that the nuclear RNA level of histones mRNAs in do not explain the high R-loop signal of these genes. It also suggests that the level of RNA polymerase II bound to histone genes does not explain the high R-loop signal observed at these genes. The bisDRIP-seq values in both plots were derived from the mean bisDRIP-seq scores of n = 13 control-treated samples and mean bisDRIP-seq scores of n = 2 triptolide-treated samples. In ( A ), we examined n = 1813 non-histone genes and n = 2 histone genes. In ( B ), we examined n = 2064 non-histone genes and n = 13 histone genes. TPKM refers to transcripts per kilobase million. The shaded areas around linear regression models represent 95% confidence intervals. ( C ) RNase H sensitivity of the bisDRIP-seq signal in histone genes. Metaplots were generated of histone gene non-template bisDRIP-seq score from three different bisDRIP-seq experiments. In the top plot, bisDRIP-seq scores were derived from ‘input’ samples (mean bisDRIP-seq score from n = 2 samples). These input samples received the entire bisDRIP-seq treatment except for the S9.6 immunoprecipitation enrichment steps. In the middle plot, bisDRIP-seq scores were derived from a bisDRIP-seq experiment that did not include an RNase H treatment (n = 1 sample). In the bottom plot, bisDRIP-seq scores were derived from an RNase H-treated bisDRIP-seq experiment (n = 1 sample). The bisDRIP-seq sample that was not treated with RNase H replicated the high non-template bisDRIP-seq scores observed in other bisDRIP-seq experiments at histone loci. On the other hand, almost no bisDRIP-seq signal was observed in either the input samples or in the bisDRIP-seq sample treated with RNase H. This suggests that the bisDRIP-seq signal observed at these gene loci is caused by R-loops. Transcription start sites are indicated by both ‘TSS’ and by dashed vertical lines. ( D,E ) bisDRIP-seq enrichment at ( D ) HIST1H2BG and ( E ) HIST1H1E is due to the presence of R-loops. In the middle and top plots, we compare the bisDRIP-seq from an RNase H treated sample (n = 1) and a bisDRIP-seq sample that was not treated with RNase H (n = 1), respectively. In both the middle and top plot, bisDRIP-seq scores from each experiment were mapped to the genomic region surrounding ( D ) HIST1H2BG and ( E ) HIST1H1E . At both loci, the bisDRIP-seq sample that was not treated with RNase H had similar bisDRIP-seq signal to our other bisDRIP-seq control experiments ( Figure 5D and E ). On the other hand, there is a clear reduction in bisDRIP-seq scores in the RNase H-treated sample. This decrease in signal after RNase H treatment suggests that RNA-DNA hybrids do form at these loci. In the bottom panel, the percentage of each nucleotide is plotted in 51 bp windows. There is no obvious relationship between nucleotide composition and bisDRIP-seq scores throughout these two genes.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Activity Assay, Derivative Assay, Generated, Immunoprecipitation

    40) Product Images from "Argonaute-based programmable RNase as a tool for cleavage of highly-structured RNA"

    Article Title: Argonaute-based programmable RNase as a tool for cleavage of highly-structured RNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky496

    Comparing cleavage activity of DISC and RNase H. ( A ) Schematic of matched and mismatched guide and target pairs used to target four TRs across the HIV-1 ΔDIS 5′UTR RNA. For each pair, the HIV-1 ΔDIS 5′UTR sequence is shown on top and the perfectly matched gDNA strand is shown on the bottom. Circle indicates target position complementary to the first position of the guide that does not pair due to structural restrains by the protein. Black arrowheads indicate cleavage site. Mismatches between the guide and target strands are indicated by a black box around the bases of the guide that are mutated to the bases shown below the box. ( B ) Quantified cleavage products from the assay using matched and mismatched guide and target pairs described in (A) are plotted with solid bars representing the average of three replicates and circles representing individual replicates. Cleavage that was not detectable by the assay is indicated by ‘nd’. ( C and D ) Comparing DISC (circles) and RNase H (triangles) cleavage of the unstructured 60-nt target (C) or of a structured 352-nt RNA target (D). Bars indicate average cleavage of three replicates.
    Figure Legend Snippet: Comparing cleavage activity of DISC and RNase H. ( A ) Schematic of matched and mismatched guide and target pairs used to target four TRs across the HIV-1 ΔDIS 5′UTR RNA. For each pair, the HIV-1 ΔDIS 5′UTR sequence is shown on top and the perfectly matched gDNA strand is shown on the bottom. Circle indicates target position complementary to the first position of the guide that does not pair due to structural restrains by the protein. Black arrowheads indicate cleavage site. Mismatches between the guide and target strands are indicated by a black box around the bases of the guide that are mutated to the bases shown below the box. ( B ) Quantified cleavage products from the assay using matched and mismatched guide and target pairs described in (A) are plotted with solid bars representing the average of three replicates and circles representing individual replicates. Cleavage that was not detectable by the assay is indicated by ‘nd’. ( C and D ) Comparing DISC (circles) and RNase H (triangles) cleavage of the unstructured 60-nt target (C) or of a structured 352-nt RNA target (D). Bars indicate average cleavage of three replicates.

    Techniques Used: Activity Assay, Sequencing

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    Article Snippet: Our data show that RNase H2 does not discriminate between canonical and oxidized ribonucleotides and can remove ribonucleotides also when these are paired with 8-oxodG.

    RNA Sequencing Assay:

    Article Title: Defining the location of promoter-associated R-loops at near-nucleotide resolution using bisDRIP-seq
    Article Snippet: .. Another possibility is that the cell may target mRNAs with retained first-introns that form R-loops to be degraded by RNase H. In this model, mRNAs with retained introns that are observable by RNA-seq must have mechanisms to prevent the RNA from forming a long R-loop that would be susceptible to RNase H degradation. ..

    Immunoprecipitation:

    Article Title: R‐loop formation during S phase is restricted by PrimPol‐mediated repriming
    Article Snippet: .. The specificity of the pull‐down was tested with RNase H and RNase III treatments prior to immunoprecipitation: one‐third of the digested material was treated with 25 U of RNase H (NEB, M0297), or with 10 U of RNase III (Ambion, AM2290) in appropriate buffers overnight at 37°C, with the subsequent steps performed as described above. ..

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    New England Biolabs rnase h
    eIF4A activity within the 48S complex. ( A ) RNase H-mapping of RNA–RNA interactions between SV-DLP U1 and 18S rRNA. The analysis was carried out in the absence or presence of 1 μM hippuristanol, with identification of the resulting RNA fragments indicated. For clarity, a schematic diagram of the ES6S and h16–18 regions of rabbit 18S rRNA with the primers used for <t>RNase</t> H digestion is shown. The use of oligos 4 and 9 limited the region of 18S rRNA (509–830) where the crosslinkings concentrated. Bands corresponding to crosslinking of SV DLP U1 mRNA with the ES6S region (680–1863) and h16-h18 helices (1–662) were quantified by densitometry and expressed as a ratio. Data are the mean ± SEM from four independent experiments. ( B ). Reactivity to SHAPE reagent (NMIA) is higher for unpaired nucleotides (red) and low for those involved in pairings (black). Stops corresponding to toeprints are marked with arrowheads. Quantification of toeprint ratios (17–19/23–25) in absence or presence of hippuristanol is shown from three independent experiments; data are the mean ± SEM.
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    eIF4A activity within the 48S complex. ( A ) RNase H-mapping of RNA–RNA interactions between SV-DLP U1 and 18S rRNA. The analysis was carried out in the absence or presence of 1 μM hippuristanol, with identification of the resulting RNA fragments indicated. For clarity, a schematic diagram of the ES6S and h16–18 regions of rabbit 18S rRNA with the primers used for RNase H digestion is shown. The use of oligos 4 and 9 limited the region of 18S rRNA (509–830) where the crosslinkings concentrated. Bands corresponding to crosslinking of SV DLP U1 mRNA with the ES6S region (680–1863) and h16-h18 helices (1–662) were quantified by densitometry and expressed as a ratio. Data are the mean ± SEM from four independent experiments. ( B ). Reactivity to SHAPE reagent (NMIA) is higher for unpaired nucleotides (red) and low for those involved in pairings (black). Stops corresponding to toeprints are marked with arrowheads. Quantification of toeprint ratios (17–19/23–25) in absence or presence of hippuristanol is shown from three independent experiments; data are the mean ± SEM.

    Journal: Nucleic Acids Research

    Article Title: Translation initiation of alphavirus mRNA reveals new insights into the topology of the 48S initiation complex

    doi: 10.1093/nar/gky071

    Figure Lengend Snippet: eIF4A activity within the 48S complex. ( A ) RNase H-mapping of RNA–RNA interactions between SV-DLP U1 and 18S rRNA. The analysis was carried out in the absence or presence of 1 μM hippuristanol, with identification of the resulting RNA fragments indicated. For clarity, a schematic diagram of the ES6S and h16–18 regions of rabbit 18S rRNA with the primers used for RNase H digestion is shown. The use of oligos 4 and 9 limited the region of 18S rRNA (509–830) where the crosslinkings concentrated. Bands corresponding to crosslinking of SV DLP U1 mRNA with the ES6S region (680–1863) and h16-h18 helices (1–662) were quantified by densitometry and expressed as a ratio. Data are the mean ± SEM from four independent experiments. ( B ). Reactivity to SHAPE reagent (NMIA) is higher for unpaired nucleotides (red) and low for those involved in pairings (black). Stops corresponding to toeprints are marked with arrowheads. Quantification of toeprint ratios (17–19/23–25) in absence or presence of hippuristanol is shown from three independent experiments; data are the mean ± SEM.

    Article Snippet: Briefly, samples were annealed at 65°C for 5′ with 10 pmol of oligonucleotides covering the indicated regions of 18S rRNA and digested with 5 U of RNase H (NEB) for 15 min at 37°C.

    Techniques: Activity Assay

    Eap1p promotes decapping of HO mRNA. (A) HO mRNA was cleaved with RNase H and a DNA oligonucleotide to produce a 1,600-nucleotide 5′ fragment and a 253-nucleotide 3′ fragment with a poly(A) tail of up to 80 adenosines (pA 80 ). (B) Northern

    Journal: Molecular and Cellular Biology

    Article Title: A Eukaryotic Translation Initiation Factor 4E-Binding Protein Promotes mRNA Decapping and Is Required for PUF Repression

    doi: 10.1128/MCB.00483-12

    Figure Lengend Snippet: Eap1p promotes decapping of HO mRNA. (A) HO mRNA was cleaved with RNase H and a DNA oligonucleotide to produce a 1,600-nucleotide 5′ fragment and a 253-nucleotide 3′ fragment with a poly(A) tail of up to 80 adenosines (pA 80 ). (B) Northern

    Article Snippet: To resolve the poly(A) tail length, HO mRNA was cleaved with RNase H and a cDNA oligonucleotide to generate a 253-nucleotide, 3′ fragment with a poly(A) tail of up to 80 nucleotides ( ).

    Techniques: Northern Blot

    bisDRIP-seq scores are sensitive to RNase H. ( A ) RNA-DNA hybrid formation is necessary for enrichment of bisDRIP-seq signal at sites of R-loop formation. RNA-DNA hybrids are integral components of R-loops. RNA-DNA hybrids are known to bind the S9.6 antibody and to be degraded by RNase H. R-loop signal should therefore be lost in the absence of S9.6 immunoprecipitation or if the sample is treated with RNase H. In each of these plots, the template strand refers to the strand used for RPPH1 transcription, rather than the template strand used for PAPR2 transcription. Also, in each plot the bisDRIP-seq score on the template strand and non-template strand were plotted below the x-axis (orange) or above the x-axis (blue), respectively. In the top plot, bisDRIP-seq scores were mapped to the genomic region surrounding RPPH1 from ‘input’ samples (mean bisDRIP-seq score from n = 2 samples). These input samples received the entire bisDRIP-seq treatment except for the S9.6 immunoprecipitation enrichment steps. In the middle plot, bisDRIP-seq scores were mapped to the genomic region surrounding RPPH1 from a control-treated bisDRIP-seq experiment that was not treated with RNase H (n = 1 sample). In the bottom plot, bisDRIP-seq scores were mapped to the genomic region surrounding RPPH1 from an RNase H-treated bisDRIP-seq experiment (n = 1 sample). The bisDRIP-seq sample that was not treated with RNase H has high bisDRIP-seq score at the RPPH1 locus. On the other hand, there is a large decrease in bisDRIP-seq signal in the sample treated with RNase H. This suggests that bisDRIP-seq signal in the RPPH1 locus depends on the presence of an RNA-DNA hybrid.

    Journal: eLife

    Article Title: Defining the location of promoter-associated R-loops at near-nucleotide resolution using bisDRIP-seq

    doi: 10.7554/eLife.28306

    Figure Lengend Snippet: bisDRIP-seq scores are sensitive to RNase H. ( A ) RNA-DNA hybrid formation is necessary for enrichment of bisDRIP-seq signal at sites of R-loop formation. RNA-DNA hybrids are integral components of R-loops. RNA-DNA hybrids are known to bind the S9.6 antibody and to be degraded by RNase H. R-loop signal should therefore be lost in the absence of S9.6 immunoprecipitation or if the sample is treated with RNase H. In each of these plots, the template strand refers to the strand used for RPPH1 transcription, rather than the template strand used for PAPR2 transcription. Also, in each plot the bisDRIP-seq score on the template strand and non-template strand were plotted below the x-axis (orange) or above the x-axis (blue), respectively. In the top plot, bisDRIP-seq scores were mapped to the genomic region surrounding RPPH1 from ‘input’ samples (mean bisDRIP-seq score from n = 2 samples). These input samples received the entire bisDRIP-seq treatment except for the S9.6 immunoprecipitation enrichment steps. In the middle plot, bisDRIP-seq scores were mapped to the genomic region surrounding RPPH1 from a control-treated bisDRIP-seq experiment that was not treated with RNase H (n = 1 sample). In the bottom plot, bisDRIP-seq scores were mapped to the genomic region surrounding RPPH1 from an RNase H-treated bisDRIP-seq experiment (n = 1 sample). The bisDRIP-seq sample that was not treated with RNase H has high bisDRIP-seq score at the RPPH1 locus. On the other hand, there is a large decrease in bisDRIP-seq signal in the sample treated with RNase H. This suggests that bisDRIP-seq signal in the RPPH1 locus depends on the presence of an RNA-DNA hybrid.

    Article Snippet: In each of these final two wash steps, RNase H was added to a final concentration of 0.5 U/µl to the ‘RNase H’ treated sample, while dithiothreitol was added to a final concentration of 50 µM to the matched control sample.

    Techniques: Immunoprecipitation

    The strand asymmetry of bisDRIP-seq scores 3' of the transcription start site is sensitive to RNase H. ( A,B ) The strand asymmetry in bisDRIP-seq scores 3' of transcription start sites is not observed after RNase H treatment. RNA-DNA hybrids are core components of R-loops. If the strand asymmetry observed in metaplots of promoter regions is due to R-loops, than it should be selectively sensitive to RNase H. We therefore generated metaplots of bisDRIP-seq scores from two samples treated identically except that ( A ) received a vehicle treatment, while ( B ) was treated with RNase H. Metaplots were created by summing the bisDRIP-seq scores across active promoter regions (n = 15644) at each nucleotide position relative to the transcription start site. bisDRIP-seq scores were calculated separately for the nucleotide position on the non-template strand (blue) and template strand (orange). In the vehicle-treated sample, non-template bisDRIP-seq scores are higher than template bisDRIP-seq scores immediately 3' of the transcription start site. This replicates the result from Figure 3E . On the other hand, there is a complete loss of bisDRIP-seq score strand asymmetry after RNase H treatment in ( B ). This confirms that the asymmetry observed in Figure 3E is caused by R-loops.

    Journal: eLife

    Article Title: Defining the location of promoter-associated R-loops at near-nucleotide resolution using bisDRIP-seq

    doi: 10.7554/eLife.28306

    Figure Lengend Snippet: The strand asymmetry of bisDRIP-seq scores 3' of the transcription start site is sensitive to RNase H. ( A,B ) The strand asymmetry in bisDRIP-seq scores 3' of transcription start sites is not observed after RNase H treatment. RNA-DNA hybrids are core components of R-loops. If the strand asymmetry observed in metaplots of promoter regions is due to R-loops, than it should be selectively sensitive to RNase H. We therefore generated metaplots of bisDRIP-seq scores from two samples treated identically except that ( A ) received a vehicle treatment, while ( B ) was treated with RNase H. Metaplots were created by summing the bisDRIP-seq scores across active promoter regions (n = 15644) at each nucleotide position relative to the transcription start site. bisDRIP-seq scores were calculated separately for the nucleotide position on the non-template strand (blue) and template strand (orange). In the vehicle-treated sample, non-template bisDRIP-seq scores are higher than template bisDRIP-seq scores immediately 3' of the transcription start site. This replicates the result from Figure 3E . On the other hand, there is a complete loss of bisDRIP-seq score strand asymmetry after RNase H treatment in ( B ). This confirms that the asymmetry observed in Figure 3E is caused by R-loops.

    Article Snippet: In each of these final two wash steps, RNase H was added to a final concentration of 0.5 U/µl to the ‘RNase H’ treated sample, while dithiothreitol was added to a final concentration of 50 µM to the matched control sample.

    Techniques: Generated

    R-loop signal in MALAT1 and NEAT1 is reduced by RNase H treatment. ( A,B ) bisDRIP-seq enrichment at ( A ) MALAT1 and ( B ) NEAT1 is lost after RNase H treatment and is not observed without RNA-DNA hybrid immunoprecipitation by S9.6 antibody. RNase H should selectively degrade the RNA component of R-loops. Thus, if bisDRIP-seq signal is caused by R-loops, RNase H treatment should reduce bisDRIP-seq signal. In the top panel, bisDRIP-seq scores from ‘input’ samples (mean of n = 2 samples) were mapped to each gene locus. bisDRIP-seq input samples were treated an identical manner to other bisDRIP-seq samples, except that no S9.6 antibody immunoprecipitation steps were performed. In the middle and lower plots, the bisDRIP-seq scores from two matched bisDRIP-seq experiments were mapped to each locus. The only difference between these experiments is that one sample was not treated with RNase H (middle plots, n = 1 sample), while the other sample was treated with RNase H (lower plots, n = 1 sample). At both loci, the sample that was not treated with RNase H replicated the bisDRIP-seq signal observed in standard bisDRIP-seq control experiments ( Figure 6A and B ). On the other hand, there is less bisDRIP-seq signal observed at each loci in both the input and RNase H-treated samples. These results suggest that RNA-DNA hybrids form in the MALAT1 and NEAT1 loci and that these RNA-DNA hybrids cause the high bisDRIP-seq scores observed at these loci. The location of the transcription start site is demarcated by ‘TSS’ and a dashed vertical line.

    Journal: eLife

    Article Title: Defining the location of promoter-associated R-loops at near-nucleotide resolution using bisDRIP-seq

    doi: 10.7554/eLife.28306

    Figure Lengend Snippet: R-loop signal in MALAT1 and NEAT1 is reduced by RNase H treatment. ( A,B ) bisDRIP-seq enrichment at ( A ) MALAT1 and ( B ) NEAT1 is lost after RNase H treatment and is not observed without RNA-DNA hybrid immunoprecipitation by S9.6 antibody. RNase H should selectively degrade the RNA component of R-loops. Thus, if bisDRIP-seq signal is caused by R-loops, RNase H treatment should reduce bisDRIP-seq signal. In the top panel, bisDRIP-seq scores from ‘input’ samples (mean of n = 2 samples) were mapped to each gene locus. bisDRIP-seq input samples were treated an identical manner to other bisDRIP-seq samples, except that no S9.6 antibody immunoprecipitation steps were performed. In the middle and lower plots, the bisDRIP-seq scores from two matched bisDRIP-seq experiments were mapped to each locus. The only difference between these experiments is that one sample was not treated with RNase H (middle plots, n = 1 sample), while the other sample was treated with RNase H (lower plots, n = 1 sample). At both loci, the sample that was not treated with RNase H replicated the bisDRIP-seq signal observed in standard bisDRIP-seq control experiments ( Figure 6A and B ). On the other hand, there is less bisDRIP-seq signal observed at each loci in both the input and RNase H-treated samples. These results suggest that RNA-DNA hybrids form in the MALAT1 and NEAT1 loci and that these RNA-DNA hybrids cause the high bisDRIP-seq scores observed at these loci. The location of the transcription start site is demarcated by ‘TSS’ and a dashed vertical line.

    Article Snippet: In each of these final two wash steps, RNase H was added to a final concentration of 0.5 U/µl to the ‘RNase H’ treated sample, while dithiothreitol was added to a final concentration of 50 µM to the matched control sample.

    Techniques: Immunoprecipitation

    Histone genes are enriched in R-loops. ( A,B ) RNA polymerase II binding and RNA levels in the nucleus do not appear to explain the association between histone genes and R-loop formation. R-loop formation was calculated for each promoter region by taking the sum of bisDRIP-seq score strand asymmetry and the triptolide-sensitivity the non-template bisDRIP-seq scores. R-loop formation was then plotted against ( A ) nuclear RNA levels or ( B ) POLR2A (RNA polymerase II) chromatin immunoprecipitation (ChIP) signal for each promoter region. Promoter regions were plotted separately for non-histone genes (blue) and replication-dependent histone genes (orange). A linear regression for all genes was also plotted (blue). Both nuclear RNA levels and RNA polymerase II ChIP signal were weakly correlated with R-loop formation. Nevertheless, replication-dependent histone genes typically had higher R-loop formation scores than genes with similar promoter activity. This suggests that the nuclear RNA level of histones mRNAs in do not explain the high R-loop signal of these genes. It also suggests that the level of RNA polymerase II bound to histone genes does not explain the high R-loop signal observed at these genes. The bisDRIP-seq values in both plots were derived from the mean bisDRIP-seq scores of n = 13 control-treated samples and mean bisDRIP-seq scores of n = 2 triptolide-treated samples. In ( A ), we examined n = 1813 non-histone genes and n = 2 histone genes. In ( B ), we examined n = 2064 non-histone genes and n = 13 histone genes. TPKM refers to transcripts per kilobase million. The shaded areas around linear regression models represent 95% confidence intervals. ( C ) RNase H sensitivity of the bisDRIP-seq signal in histone genes. Metaplots were generated of histone gene non-template bisDRIP-seq score from three different bisDRIP-seq experiments. In the top plot, bisDRIP-seq scores were derived from ‘input’ samples (mean bisDRIP-seq score from n = 2 samples). These input samples received the entire bisDRIP-seq treatment except for the S9.6 immunoprecipitation enrichment steps. In the middle plot, bisDRIP-seq scores were derived from a bisDRIP-seq experiment that did not include an RNase H treatment (n = 1 sample). In the bottom plot, bisDRIP-seq scores were derived from an RNase H-treated bisDRIP-seq experiment (n = 1 sample). The bisDRIP-seq sample that was not treated with RNase H replicated the high non-template bisDRIP-seq scores observed in other bisDRIP-seq experiments at histone loci. On the other hand, almost no bisDRIP-seq signal was observed in either the input samples or in the bisDRIP-seq sample treated with RNase H. This suggests that the bisDRIP-seq signal observed at these gene loci is caused by R-loops. Transcription start sites are indicated by both ‘TSS’ and by dashed vertical lines. ( D,E ) bisDRIP-seq enrichment at ( D ) HIST1H2BG and ( E ) HIST1H1E is due to the presence of R-loops. In the middle and top plots, we compare the bisDRIP-seq from an RNase H treated sample (n = 1) and a bisDRIP-seq sample that was not treated with RNase H (n = 1), respectively. In both the middle and top plot, bisDRIP-seq scores from each experiment were mapped to the genomic region surrounding ( D ) HIST1H2BG and ( E ) HIST1H1E . At both loci, the bisDRIP-seq sample that was not treated with RNase H had similar bisDRIP-seq signal to our other bisDRIP-seq control experiments ( Figure 5D and E ). On the other hand, there is a clear reduction in bisDRIP-seq scores in the RNase H-treated sample. This decrease in signal after RNase H treatment suggests that RNA-DNA hybrids do form at these loci. In the bottom panel, the percentage of each nucleotide is plotted in 51 bp windows. There is no obvious relationship between nucleotide composition and bisDRIP-seq scores throughout these two genes.

    Journal: eLife

    Article Title: Defining the location of promoter-associated R-loops at near-nucleotide resolution using bisDRIP-seq

    doi: 10.7554/eLife.28306

    Figure Lengend Snippet: Histone genes are enriched in R-loops. ( A,B ) RNA polymerase II binding and RNA levels in the nucleus do not appear to explain the association between histone genes and R-loop formation. R-loop formation was calculated for each promoter region by taking the sum of bisDRIP-seq score strand asymmetry and the triptolide-sensitivity the non-template bisDRIP-seq scores. R-loop formation was then plotted against ( A ) nuclear RNA levels or ( B ) POLR2A (RNA polymerase II) chromatin immunoprecipitation (ChIP) signal for each promoter region. Promoter regions were plotted separately for non-histone genes (blue) and replication-dependent histone genes (orange). A linear regression for all genes was also plotted (blue). Both nuclear RNA levels and RNA polymerase II ChIP signal were weakly correlated with R-loop formation. Nevertheless, replication-dependent histone genes typically had higher R-loop formation scores than genes with similar promoter activity. This suggests that the nuclear RNA level of histones mRNAs in do not explain the high R-loop signal of these genes. It also suggests that the level of RNA polymerase II bound to histone genes does not explain the high R-loop signal observed at these genes. The bisDRIP-seq values in both plots were derived from the mean bisDRIP-seq scores of n = 13 control-treated samples and mean bisDRIP-seq scores of n = 2 triptolide-treated samples. In ( A ), we examined n = 1813 non-histone genes and n = 2 histone genes. In ( B ), we examined n = 2064 non-histone genes and n = 13 histone genes. TPKM refers to transcripts per kilobase million. The shaded areas around linear regression models represent 95% confidence intervals. ( C ) RNase H sensitivity of the bisDRIP-seq signal in histone genes. Metaplots were generated of histone gene non-template bisDRIP-seq score from three different bisDRIP-seq experiments. In the top plot, bisDRIP-seq scores were derived from ‘input’ samples (mean bisDRIP-seq score from n = 2 samples). These input samples received the entire bisDRIP-seq treatment except for the S9.6 immunoprecipitation enrichment steps. In the middle plot, bisDRIP-seq scores were derived from a bisDRIP-seq experiment that did not include an RNase H treatment (n = 1 sample). In the bottom plot, bisDRIP-seq scores were derived from an RNase H-treated bisDRIP-seq experiment (n = 1 sample). The bisDRIP-seq sample that was not treated with RNase H replicated the high non-template bisDRIP-seq scores observed in other bisDRIP-seq experiments at histone loci. On the other hand, almost no bisDRIP-seq signal was observed in either the input samples or in the bisDRIP-seq sample treated with RNase H. This suggests that the bisDRIP-seq signal observed at these gene loci is caused by R-loops. Transcription start sites are indicated by both ‘TSS’ and by dashed vertical lines. ( D,E ) bisDRIP-seq enrichment at ( D ) HIST1H2BG and ( E ) HIST1H1E is due to the presence of R-loops. In the middle and top plots, we compare the bisDRIP-seq from an RNase H treated sample (n = 1) and a bisDRIP-seq sample that was not treated with RNase H (n = 1), respectively. In both the middle and top plot, bisDRIP-seq scores from each experiment were mapped to the genomic region surrounding ( D ) HIST1H2BG and ( E ) HIST1H1E . At both loci, the bisDRIP-seq sample that was not treated with RNase H had similar bisDRIP-seq signal to our other bisDRIP-seq control experiments ( Figure 5D and E ). On the other hand, there is a clear reduction in bisDRIP-seq scores in the RNase H-treated sample. This decrease in signal after RNase H treatment suggests that RNA-DNA hybrids do form at these loci. In the bottom panel, the percentage of each nucleotide is plotted in 51 bp windows. There is no obvious relationship between nucleotide composition and bisDRIP-seq scores throughout these two genes.

    Article Snippet: In each of these final two wash steps, RNase H was added to a final concentration of 0.5 U/µl to the ‘RNase H’ treated sample, while dithiothreitol was added to a final concentration of 50 µM to the matched control sample.

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Activity Assay, Derivative Assay, Generated, Immunoprecipitation