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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 generated by RNA Polymerase III are substrates for cellular RNase H.A: Analysis of R-loops by ChIP-QPCR using antibody S9.6 in wild-type strain BY4741 (WT) and double mutant rnh1Δ rnh201Δ, and in triple mutant PGAL-TOP1 rnh1Δ rnh201Δ depleted of Top1 for 6h at 30°C. CEN16, the Pol II genes ADH1 and ACT1, the Pol I transcribed 35S rRNA gene, and the Pol III genes (5S rDNA, tRNA tQ(UUG)L, SCR1, SNR6, tRNA tS(GCU)F and tRNA SUF2) were analyzed by Q-PCR. Values for no-antibody (−Ab) and antibody S9.6 (+Ab) were calculated as described in Materials and Methods and normalized to CEN16, which was set arbitrarily to 1 in order to compensate for differences in immunoprecipitation efficiencies. CEN16 is not expected to be transcribed and should therefore give rise only to background signal after immunoprecipitation. The mean of three independent experiments is shown with standard error. B: S9.6-ChIP samples of strains WT (BY4741) and double mutant rnh1Δ rnh201Δ, grown at 30°C in YEPD (glucose 2%), were treated or not with recombinant RNase H ‘on-beads’ for 2.5 h at 37°C. CEN16, 18S rDNA, mitochondrial 21S rDNA, Pol III genes [same as in (A)] and Ty1 retrotransposons, were analyzed by Q-PCR as described above. rnhΔΔ = double mutant rnh1Δ rnh201Δ. C–D: Heatmaps of R-loop distribution across 274 tRNA genes assigned to 41 families of distinct codon specificity in the WT (C) and double mutant rnh1Δ rnh201Δ (D). Genes are ordered by their anticodon rank (Y-axis) based on the sum of codons in the genome for which it can be used, with a value of 1 representing the anticodon with the highest number of codons in the genome. Anticodons were grouped into 41 families (see [99]), and the codon frequencies were calculated from all protein coding genes in the Saccer3 genome assembly. The X-axis shows the position of the tRNA genes with 1 kb of 5′- and 3′- flanking sequences. Each point on the graph is a colored tile representing the fold change of: “[WT S9.6 ChIP-seq] relative to [input chromatin]” panel (C); and “[rnh1Δ rnh201Δ S9.6 ChIP-seq] relative to [WT S9.6 ChIP-seq]” panel (D). 5′ and 3′ endpoints of mature tRNAs are delineated by vertical dotted lines across the heatmaps.
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pgen-1004716-g001: R-loops generated by RNA Polymerase III are substrates for cellular RNase H.A: Analysis of R-loops by ChIP-QPCR using antibody S9.6 in wild-type strain BY4741 (WT) and double mutant rnh1Δ rnh201Δ, and in triple mutant PGAL-TOP1 rnh1Δ rnh201Δ depleted of Top1 for 6h at 30°C. CEN16, the Pol II genes ADH1 and ACT1, the Pol I transcribed 35S rRNA gene, and the Pol III genes (5S rDNA, tRNA tQ(UUG)L, SCR1, SNR6, tRNA tS(GCU)F and tRNA SUF2) were analyzed by Q-PCR. Values for no-antibody (−Ab) and antibody S9.6 (+Ab) were calculated as described in Materials and Methods and normalized to CEN16, which was set arbitrarily to 1 in order to compensate for differences in immunoprecipitation efficiencies. CEN16 is not expected to be transcribed and should therefore give rise only to background signal after immunoprecipitation. The mean of three independent experiments is shown with standard error. B: S9.6-ChIP samples of strains WT (BY4741) and double mutant rnh1Δ rnh201Δ, grown at 30°C in YEPD (glucose 2%), were treated or not with recombinant RNase H ‘on-beads’ for 2.5 h at 37°C. CEN16, 18S rDNA, mitochondrial 21S rDNA, Pol III genes [same as in (A)] and Ty1 retrotransposons, were analyzed by Q-PCR as described above. rnhΔΔ = double mutant rnh1Δ rnh201Δ. C–D: Heatmaps of R-loop distribution across 274 tRNA genes assigned to 41 families of distinct codon specificity in the WT (C) and double mutant rnh1Δ rnh201Δ (D). Genes are ordered by their anticodon rank (Y-axis) based on the sum of codons in the genome for which it can be used, with a value of 1 representing the anticodon with the highest number of codons in the genome. Anticodons were grouped into 41 families (see [99]), and the codon frequencies were calculated from all protein coding genes in the Saccer3 genome assembly. The X-axis shows the position of the tRNA genes with 1 kb of 5′- and 3′- flanking sequences. Each point on the graph is a colored tile representing the fold change of: “[WT S9.6 ChIP-seq] relative to [input chromatin]” panel (C); and “[rnh1Δ rnh201Δ S9.6 ChIP-seq] relative to [WT S9.6 ChIP-seq]” panel (D). 5′ and 3′ endpoints of mature tRNAs are delineated by vertical dotted lines across the heatmaps.

Mentions: ChIP-seq was applied to wild-type and to mutants double rnh1Δ rnh201Δ and triple PGAL::TOP1 rnh1Δ rnh201Δ (Fig. S1). For Top1 depletion, cells were shifted from medium containing galactose plus sucrose and harvested after 6 h in glucose-containing medium. Sequenced reads over each target were normalized to the genome-wide mean of all intergenic regions (arbitrarily set as sequencing background, see Materials and Methods), so changes in hit densities are relative differences compared to all other targets. Reads mapped to the rDNA in strains WT, rnh1Δ rnh201Δ and PGAL::TOP1 rnh1Δ rnh201Δ (depleted of Top1), were greatly enriched in the S9.6 ChIP-seq data over the input chromatin (Fig. S1B). This is a good indication that R-loops are strongly associated with this locus, as also observed in S9.6 ChIP-QPCR (Figs. 1A–B; and [23]). For the Pol I transcribed, 35S pre-rRNA region of the rDNA, the strongest peak detected in the wild-type strain was located over the 5′ segment of the 18S rDNA (region ∼210 nt to ∼580 nt at the beginning of 18S rRNA; triple asterisk in Fig. S1A). Additional peaks were located over the 25S rDNA (e.g. quadruple asterisk in Fig. S1A). In strains lacking both cellular RNase H and Top1 a new peak appeared over the Pol I promoter and 5′ETS regions at the 5′ end of the 35S pre-rRNA (double asterisk in Fig. S1A; see also ChIP-QPCR in Fig. 1A). A further prominent peak was seen over the 5S rDNA, which is transcribed by RNA Pol III in the opposite direction to the 35S pre-rRNA (single asterisk in Fig. S1A). Notably, R-loops over the 5S rDNA were strongly increased in strains lacking RNase H and even more when Top1 was also absent (single asterisk in Fig. S1A; see also ChIP-QPCR in Fig. 1A). Comparison to DNA base-composition indicated that the uneven distribution of R-loops over the transcribed regions of the rDNA partially reflects a preference for C+G rich sequences (Fig. S1A).


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 generated by RNA Polymerase III are substrates for cellular RNase H.A: Analysis of R-loops by ChIP-QPCR using antibody S9.6 in wild-type strain BY4741 (WT) and double mutant rnh1Δ rnh201Δ, and in triple mutant PGAL-TOP1 rnh1Δ rnh201Δ depleted of Top1 for 6h at 30°C. CEN16, the Pol II genes ADH1 and ACT1, the Pol I transcribed 35S rRNA gene, and the Pol III genes (5S rDNA, tRNA tQ(UUG)L, SCR1, SNR6, tRNA tS(GCU)F and tRNA SUF2) were analyzed by Q-PCR. Values for no-antibody (−Ab) and antibody S9.6 (+Ab) were calculated as described in Materials and Methods and normalized to CEN16, which was set arbitrarily to 1 in order to compensate for differences in immunoprecipitation efficiencies. CEN16 is not expected to be transcribed and should therefore give rise only to background signal after immunoprecipitation. The mean of three independent experiments is shown with standard error. B: S9.6-ChIP samples of strains WT (BY4741) and double mutant rnh1Δ rnh201Δ, grown at 30°C in YEPD (glucose 2%), were treated or not with recombinant RNase H ‘on-beads’ for 2.5 h at 37°C. CEN16, 18S rDNA, mitochondrial 21S rDNA, Pol III genes [same as in (A)] and Ty1 retrotransposons, were analyzed by Q-PCR as described above. rnhΔΔ = double mutant rnh1Δ rnh201Δ. C–D: Heatmaps of R-loop distribution across 274 tRNA genes assigned to 41 families of distinct codon specificity in the WT (C) and double mutant rnh1Δ rnh201Δ (D). Genes are ordered by their anticodon rank (Y-axis) based on the sum of codons in the genome for which it can be used, with a value of 1 representing the anticodon with the highest number of codons in the genome. Anticodons were grouped into 41 families (see [99]), and the codon frequencies were calculated from all protein coding genes in the Saccer3 genome assembly. The X-axis shows the position of the tRNA genes with 1 kb of 5′- and 3′- flanking sequences. Each point on the graph is a colored tile representing the fold change of: “[WT S9.6 ChIP-seq] relative to [input chromatin]” panel (C); and “[rnh1Δ rnh201Δ S9.6 ChIP-seq] relative to [WT S9.6 ChIP-seq]” panel (D). 5′ and 3′ endpoints of mature tRNAs are delineated by vertical dotted lines across the heatmaps.
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Related In: Results  -  Collection

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Show All Figures
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pgen-1004716-g001: R-loops generated by RNA Polymerase III are substrates for cellular RNase H.A: Analysis of R-loops by ChIP-QPCR using antibody S9.6 in wild-type strain BY4741 (WT) and double mutant rnh1Δ rnh201Δ, and in triple mutant PGAL-TOP1 rnh1Δ rnh201Δ depleted of Top1 for 6h at 30°C. CEN16, the Pol II genes ADH1 and ACT1, the Pol I transcribed 35S rRNA gene, and the Pol III genes (5S rDNA, tRNA tQ(UUG)L, SCR1, SNR6, tRNA tS(GCU)F and tRNA SUF2) were analyzed by Q-PCR. Values for no-antibody (−Ab) and antibody S9.6 (+Ab) were calculated as described in Materials and Methods and normalized to CEN16, which was set arbitrarily to 1 in order to compensate for differences in immunoprecipitation efficiencies. CEN16 is not expected to be transcribed and should therefore give rise only to background signal after immunoprecipitation. The mean of three independent experiments is shown with standard error. B: S9.6-ChIP samples of strains WT (BY4741) and double mutant rnh1Δ rnh201Δ, grown at 30°C in YEPD (glucose 2%), were treated or not with recombinant RNase H ‘on-beads’ for 2.5 h at 37°C. CEN16, 18S rDNA, mitochondrial 21S rDNA, Pol III genes [same as in (A)] and Ty1 retrotransposons, were analyzed by Q-PCR as described above. rnhΔΔ = double mutant rnh1Δ rnh201Δ. C–D: Heatmaps of R-loop distribution across 274 tRNA genes assigned to 41 families of distinct codon specificity in the WT (C) and double mutant rnh1Δ rnh201Δ (D). Genes are ordered by their anticodon rank (Y-axis) based on the sum of codons in the genome for which it can be used, with a value of 1 representing the anticodon with the highest number of codons in the genome. Anticodons were grouped into 41 families (see [99]), and the codon frequencies were calculated from all protein coding genes in the Saccer3 genome assembly. The X-axis shows the position of the tRNA genes with 1 kb of 5′- and 3′- flanking sequences. Each point on the graph is a colored tile representing the fold change of: “[WT S9.6 ChIP-seq] relative to [input chromatin]” panel (C); and “[rnh1Δ rnh201Δ S9.6 ChIP-seq] relative to [WT S9.6 ChIP-seq]” panel (D). 5′ and 3′ endpoints of mature tRNAs are delineated by vertical dotted lines across the heatmaps.
Mentions: ChIP-seq was applied to wild-type and to mutants double rnh1Δ rnh201Δ and triple PGAL::TOP1 rnh1Δ rnh201Δ (Fig. S1). For Top1 depletion, cells were shifted from medium containing galactose plus sucrose and harvested after 6 h in glucose-containing medium. Sequenced reads over each target were normalized to the genome-wide mean of all intergenic regions (arbitrarily set as sequencing background, see Materials and Methods), so changes in hit densities are relative differences compared to all other targets. Reads mapped to the rDNA in strains WT, rnh1Δ rnh201Δ and PGAL::TOP1 rnh1Δ rnh201Δ (depleted of Top1), were greatly enriched in the S9.6 ChIP-seq data over the input chromatin (Fig. S1B). This is a good indication that R-loops are strongly associated with this locus, as also observed in S9.6 ChIP-QPCR (Figs. 1A–B; and [23]). For the Pol I transcribed, 35S pre-rRNA region of the rDNA, the strongest peak detected in the wild-type strain was located over the 5′ segment of the 18S rDNA (region ∼210 nt to ∼580 nt at the beginning of 18S rRNA; triple asterisk in Fig. S1A). Additional peaks were located over the 25S rDNA (e.g. quadruple asterisk in Fig. S1A). In strains lacking both cellular RNase H and Top1 a new peak appeared over the Pol I promoter and 5′ETS regions at the 5′ end of the 35S pre-rRNA (double asterisk in Fig. S1A; see also ChIP-QPCR in Fig. 1A). A further prominent peak was seen over the 5S rDNA, which is transcribed by RNA Pol III in the opposite direction to the 35S pre-rRNA (single asterisk in Fig. S1A). Notably, R-loops over the 5S rDNA were strongly increased in strains lacking RNase H and even more when Top1 was also absent (single asterisk in Fig. S1A; see also ChIP-QPCR in Fig. 1A). Comparison to DNA base-composition indicated that the uneven distribution of R-loops over the transcribed regions of the rDNA partially reflects a preference for C+G rich sequences (Fig. S1A).

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