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Genome-wide distribution of RNA-DNA hybrids identifies RNase H targets in tRNA genes, retrotransposons and mitochondria.

El Hage A, Webb S, Kerr A, Tollervey D - PLoS Genet. (2014)

Bottom Line: In wild-type strains, R-loops were readily detected over the 35S rDNA region, transcribed by Pol I, and over the 5S rDNA, transcribed by Pol III.In strains lacking RNase H activity, R-loops were elevated over other Pol III genes, notably tRNAs, SCR1 and U6 snRNA, and were also associated with the cDNAs of endogenous TY1 retrotransposons, which showed increased rates of mobility to the 5'-flanking regions of tRNA genes.Finally, R-loops were detected on actively transcribed protein-coding genes in the wild-type, particularly over the second exon of spliced ribosomal protein genes.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, United Kingdom.

ABSTRACT
During transcription, the nascent RNA can invade the DNA template, forming extended RNA-DNA duplexes (R-loops). Here we employ ChIP-seq in strains expressing or lacking RNase H to map targets of RNase H activity throughout the budding yeast genome. In wild-type strains, R-loops were readily detected over the 35S rDNA region, transcribed by Pol I, and over the 5S rDNA, transcribed by Pol III. In strains lacking RNase H activity, R-loops were elevated over other Pol III genes, notably tRNAs, SCR1 and U6 snRNA, and were also associated with the cDNAs of endogenous TY1 retrotransposons, which showed increased rates of mobility to the 5'-flanking regions of tRNA genes. Unexpectedly, R-loops were also associated with mitochondrial genes in the absence of RNase H1, but not of RNase H2. Finally, R-loops were detected on actively transcribed protein-coding genes in the wild-type, particularly over the second exon of spliced ribosomal protein genes.

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R-loops are enriched over the second exon of intron-containing genes.A–B: Box plots of mean sequence read distribution per gene across all yeast protein-coding genes (n = 5864, see Fig. S10A) in samples “input chromatin” and “S9.6 ChIP-seq” from the wild-type strain (BY4741) grown at 30°C in YEPD medium (glucose 2%). The y-axis represents the relative enrichment of sequencing reads where values >1 are above the background level of sequencing (i.e. general intergenic mean, see Materials and Methods). (A) Genes were clustered arbitrarily in 6 main categories based on the strength of their mRNA expression (X-axis): C1-0 (very low), C1 (low), C2 (medium-low), C3 (medium-high), C4 (high) and C4-max (very high) (see Fig. S10A and [101]). We used a Kolmogorov-Smirnov test to determine whether levels of mean sequence distribution differ significantly between the input chromatin and ChIP-seq data within each mRNA expression group: C1-0 (D = 0.5, p-value = 1.85E-008), C1 (D = 0.8109685, p-value = 0), C2 (D = 0.8624161, p-value = 0), C3 (D = 0.8607383, p-value = 0), C4 (D = 0.8439024, p-value = 0) and C4-max (D = 0.7888889, p-value = 4.440892E-16). (B) Genes were clustered based on their GC composition across the entire length of the gene (X-axis). Box-plots show median values (black line) +/−25% quartiles in the box and minimum/maximum distribution of the values (excluding outliers) in the whiskers. The width of the boxes reflects the number of genes in each group. C: ChIP-QPCR analysis of R-loops over control CEN16 and mRNA genes ADH1, ACT1 and intron-gene RPL28 (CYH2) in strain wild-type (BY4741), grown at 30°C in YEPD medium (glucose 2%). Q-PCR values were calculated and normalized to CEN16 as in Fig. 1A. The mean of three independent experiments is shown with standard error. Ab = antibody S9.6. Exon1 and intron regions of RPL28 are represented with a filled box and a horizontal line, respectively. D–F: Box plots of S9.6 ChIP-seq profiles of R-loops over mRNA genes in the wild-type (BY4741) (same samples as in panel A). (D) Intron-containing, non-ribosomal protein genes (NRPG i-genes). (E) Intron-containing, ribosomal protein genes (RPG i-genes). (F) Top 387 highly expressed intronless genes (e-genes). Each box plot represents the log2 fold-change of S9.6 ChIP-seq relative to input chromatin, so the regions above zero value on the Y-axis are enriched with R-loops (see also Fig. S13). For ease of comparison between panels the horizontal dotted green line points to the position of the top R-loop signal on exon 2 in panel E. For i-genes in panels D-E, R-loop profiles are plotted across the Exon1-Intron-Exon2 region. The 5′ end of Exon 1 is defined either as the AUG start codon, or 100 bp upstream of the 5′ splice site for genes with Exon 1 <100 pb (see also Protocol S1). For e-genes in panel F (top 387 highly expressed, see Fig. S10A), R-loop profiles are plotted across the entire length of the gene.
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pgen-1004716-g005: R-loops are enriched over the second exon of intron-containing genes.A–B: Box plots of mean sequence read distribution per gene across all yeast protein-coding genes (n = 5864, see Fig. S10A) in samples “input chromatin” and “S9.6 ChIP-seq” from the wild-type strain (BY4741) grown at 30°C in YEPD medium (glucose 2%). The y-axis represents the relative enrichment of sequencing reads where values >1 are above the background level of sequencing (i.e. general intergenic mean, see Materials and Methods). (A) Genes were clustered arbitrarily in 6 main categories based on the strength of their mRNA expression (X-axis): C1-0 (very low), C1 (low), C2 (medium-low), C3 (medium-high), C4 (high) and C4-max (very high) (see Fig. S10A and [101]). We used a Kolmogorov-Smirnov test to determine whether levels of mean sequence distribution differ significantly between the input chromatin and ChIP-seq data within each mRNA expression group: C1-0 (D = 0.5, p-value = 1.85E-008), C1 (D = 0.8109685, p-value = 0), C2 (D = 0.8624161, p-value = 0), C3 (D = 0.8607383, p-value = 0), C4 (D = 0.8439024, p-value = 0) and C4-max (D = 0.7888889, p-value = 4.440892E-16). (B) Genes were clustered based on their GC composition across the entire length of the gene (X-axis). Box-plots show median values (black line) +/−25% quartiles in the box and minimum/maximum distribution of the values (excluding outliers) in the whiskers. The width of the boxes reflects the number of genes in each group. C: ChIP-QPCR analysis of R-loops over control CEN16 and mRNA genes ADH1, ACT1 and intron-gene RPL28 (CYH2) in strain wild-type (BY4741), grown at 30°C in YEPD medium (glucose 2%). Q-PCR values were calculated and normalized to CEN16 as in Fig. 1A. The mean of three independent experiments is shown with standard error. Ab = antibody S9.6. Exon1 and intron regions of RPL28 are represented with a filled box and a horizontal line, respectively. D–F: Box plots of S9.6 ChIP-seq profiles of R-loops over mRNA genes in the wild-type (BY4741) (same samples as in panel A). (D) Intron-containing, non-ribosomal protein genes (NRPG i-genes). (E) Intron-containing, ribosomal protein genes (RPG i-genes). (F) Top 387 highly expressed intronless genes (e-genes). Each box plot represents the log2 fold-change of S9.6 ChIP-seq relative to input chromatin, so the regions above zero value on the Y-axis are enriched with R-loops (see also Fig. S13). For ease of comparison between panels the horizontal dotted green line points to the position of the top R-loop signal on exon 2 in panel E. For i-genes in panels D-E, R-loop profiles are plotted across the Exon1-Intron-Exon2 region. The 5′ end of Exon 1 is defined either as the AUG start codon, or 100 bp upstream of the 5′ splice site for genes with Exon 1 <100 pb (see also Protocol S1). For e-genes in panel F (top 387 highly expressed, see Fig. S10A), R-loop profiles are plotted across the entire length of the gene.

Mentions: In the wild-type strain, clear enrichment for R-loops in the S9.6 ChIP-seq data relative to the input chromatin was seen at highly expressed mRNA genes (Fig. 5A). Most mRNA genes showing clear enrichment for R-loops also have relatively high G.C contents (Fig. 5B). The ChIP-seq findings were confirmed by ChIP-QPCR for the highly expressed genes ADH1, ACT1 and RPL28, which showed a small but significant enrichment in R-loops (+Ab red bars) relative to no-antibody control (−Ab black bars) (Fig. 5C; see also gene PMA1 in Fig. S9).


Genome-wide distribution of RNA-DNA hybrids identifies RNase H targets in tRNA genes, retrotransposons and mitochondria.

El Hage A, Webb S, Kerr A, Tollervey D - PLoS Genet. (2014)

R-loops are enriched over the second exon of intron-containing genes.A–B: Box plots of mean sequence read distribution per gene across all yeast protein-coding genes (n = 5864, see Fig. S10A) in samples “input chromatin” and “S9.6 ChIP-seq” from the wild-type strain (BY4741) grown at 30°C in YEPD medium (glucose 2%). The y-axis represents the relative enrichment of sequencing reads where values >1 are above the background level of sequencing (i.e. general intergenic mean, see Materials and Methods). (A) Genes were clustered arbitrarily in 6 main categories based on the strength of their mRNA expression (X-axis): C1-0 (very low), C1 (low), C2 (medium-low), C3 (medium-high), C4 (high) and C4-max (very high) (see Fig. S10A and [101]). We used a Kolmogorov-Smirnov test to determine whether levels of mean sequence distribution differ significantly between the input chromatin and ChIP-seq data within each mRNA expression group: C1-0 (D = 0.5, p-value = 1.85E-008), C1 (D = 0.8109685, p-value = 0), C2 (D = 0.8624161, p-value = 0), C3 (D = 0.8607383, p-value = 0), C4 (D = 0.8439024, p-value = 0) and C4-max (D = 0.7888889, p-value = 4.440892E-16). (B) Genes were clustered based on their GC composition across the entire length of the gene (X-axis). Box-plots show median values (black line) +/−25% quartiles in the box and minimum/maximum distribution of the values (excluding outliers) in the whiskers. The width of the boxes reflects the number of genes in each group. C: ChIP-QPCR analysis of R-loops over control CEN16 and mRNA genes ADH1, ACT1 and intron-gene RPL28 (CYH2) in strain wild-type (BY4741), grown at 30°C in YEPD medium (glucose 2%). Q-PCR values were calculated and normalized to CEN16 as in Fig. 1A. The mean of three independent experiments is shown with standard error. Ab = antibody S9.6. Exon1 and intron regions of RPL28 are represented with a filled box and a horizontal line, respectively. D–F: Box plots of S9.6 ChIP-seq profiles of R-loops over mRNA genes in the wild-type (BY4741) (same samples as in panel A). (D) Intron-containing, non-ribosomal protein genes (NRPG i-genes). (E) Intron-containing, ribosomal protein genes (RPG i-genes). (F) Top 387 highly expressed intronless genes (e-genes). Each box plot represents the log2 fold-change of S9.6 ChIP-seq relative to input chromatin, so the regions above zero value on the Y-axis are enriched with R-loops (see also Fig. S13). For ease of comparison between panels the horizontal dotted green line points to the position of the top R-loop signal on exon 2 in panel E. For i-genes in panels D-E, R-loop profiles are plotted across the Exon1-Intron-Exon2 region. The 5′ end of Exon 1 is defined either as the AUG start codon, or 100 bp upstream of the 5′ splice site for genes with Exon 1 <100 pb (see also Protocol S1). For e-genes in panel F (top 387 highly expressed, see Fig. S10A), R-loop profiles are plotted across the entire length of the gene.
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Show All Figures
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pgen-1004716-g005: R-loops are enriched over the second exon of intron-containing genes.A–B: Box plots of mean sequence read distribution per gene across all yeast protein-coding genes (n = 5864, see Fig. S10A) in samples “input chromatin” and “S9.6 ChIP-seq” from the wild-type strain (BY4741) grown at 30°C in YEPD medium (glucose 2%). The y-axis represents the relative enrichment of sequencing reads where values >1 are above the background level of sequencing (i.e. general intergenic mean, see Materials and Methods). (A) Genes were clustered arbitrarily in 6 main categories based on the strength of their mRNA expression (X-axis): C1-0 (very low), C1 (low), C2 (medium-low), C3 (medium-high), C4 (high) and C4-max (very high) (see Fig. S10A and [101]). We used a Kolmogorov-Smirnov test to determine whether levels of mean sequence distribution differ significantly between the input chromatin and ChIP-seq data within each mRNA expression group: C1-0 (D = 0.5, p-value = 1.85E-008), C1 (D = 0.8109685, p-value = 0), C2 (D = 0.8624161, p-value = 0), C3 (D = 0.8607383, p-value = 0), C4 (D = 0.8439024, p-value = 0) and C4-max (D = 0.7888889, p-value = 4.440892E-16). (B) Genes were clustered based on their GC composition across the entire length of the gene (X-axis). Box-plots show median values (black line) +/−25% quartiles in the box and minimum/maximum distribution of the values (excluding outliers) in the whiskers. The width of the boxes reflects the number of genes in each group. C: ChIP-QPCR analysis of R-loops over control CEN16 and mRNA genes ADH1, ACT1 and intron-gene RPL28 (CYH2) in strain wild-type (BY4741), grown at 30°C in YEPD medium (glucose 2%). Q-PCR values were calculated and normalized to CEN16 as in Fig. 1A. The mean of three independent experiments is shown with standard error. Ab = antibody S9.6. Exon1 and intron regions of RPL28 are represented with a filled box and a horizontal line, respectively. D–F: Box plots of S9.6 ChIP-seq profiles of R-loops over mRNA genes in the wild-type (BY4741) (same samples as in panel A). (D) Intron-containing, non-ribosomal protein genes (NRPG i-genes). (E) Intron-containing, ribosomal protein genes (RPG i-genes). (F) Top 387 highly expressed intronless genes (e-genes). Each box plot represents the log2 fold-change of S9.6 ChIP-seq relative to input chromatin, so the regions above zero value on the Y-axis are enriched with R-loops (see also Fig. S13). For ease of comparison between panels the horizontal dotted green line points to the position of the top R-loop signal on exon 2 in panel E. For i-genes in panels D-E, R-loop profiles are plotted across the Exon1-Intron-Exon2 region. The 5′ end of Exon 1 is defined either as the AUG start codon, or 100 bp upstream of the 5′ splice site for genes with Exon 1 <100 pb (see also Protocol S1). For e-genes in panel F (top 387 highly expressed, see Fig. S10A), R-loop profiles are plotted across the entire length of the gene.
Mentions: In the wild-type strain, clear enrichment for R-loops in the S9.6 ChIP-seq data relative to the input chromatin was seen at highly expressed mRNA genes (Fig. 5A). Most mRNA genes showing clear enrichment for R-loops also have relatively high G.C contents (Fig. 5B). The ChIP-seq findings were confirmed by ChIP-QPCR for the highly expressed genes ADH1, ACT1 and RPL28, which showed a small but significant enrichment in R-loops (+Ab red bars) relative to no-antibody control (−Ab black bars) (Fig. 5C; see also gene PMA1 in Fig. S9).

Bottom Line: In wild-type strains, R-loops were readily detected over the 35S rDNA region, transcribed by Pol I, and over the 5S rDNA, transcribed by Pol III.In strains lacking RNase H activity, R-loops were elevated over other Pol III genes, notably tRNAs, SCR1 and U6 snRNA, and were also associated with the cDNAs of endogenous TY1 retrotransposons, which showed increased rates of mobility to the 5'-flanking regions of tRNA genes.Finally, R-loops were detected on actively transcribed protein-coding genes in the wild-type, particularly over the second exon of spliced ribosomal protein genes.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, United Kingdom.

ABSTRACT
During transcription, the nascent RNA can invade the DNA template, forming extended RNA-DNA duplexes (R-loops). Here we employ ChIP-seq in strains expressing or lacking RNase H to map targets of RNase H activity throughout the budding yeast genome. In wild-type strains, R-loops were readily detected over the 35S rDNA region, transcribed by Pol I, and over the 5S rDNA, transcribed by Pol III. In strains lacking RNase H activity, R-loops were elevated over other Pol III genes, notably tRNAs, SCR1 and U6 snRNA, and were also associated with the cDNAs of endogenous TY1 retrotransposons, which showed increased rates of mobility to the 5'-flanking regions of tRNA genes. Unexpectedly, R-loops were also associated with mitochondrial genes in the absence of RNase H1, but not of RNase H2. Finally, R-loops were detected on actively transcribed protein-coding genes in the wild-type, particularly over the second exon of spliced ribosomal protein genes.

Show MeSH
Related in: MedlinePlus