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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 at tRNA genes affect pre-tRNA synthesis in strains lacking topoisomerase and RNase H activities.A–E: Wild-type strain BY4741 (WT) and isogenic mutant strains single PGAL-TOP1, triple PGAL-TOP1 rnh1Δ rnh201Δ, double PGAL-TOP1/TOP2 and quadruple PGAL-TOP1/TOP2 rnh1Δ rnh201Δ, were grown at 30°C and harvested at 0 h (galactose- and sucrose- containing medium) and at 6–9 h post-shift to glucose-containing medium. Total RNAs were extracted and analyzed by northern hybridization. The membrane was hybridized separately with probes tRNAiMET (A), tRNA3LEU (B), tRNA2LYS (C) and tRNATRP (D). Ethidium bromide staining of 5S rRNA is in (E). Precursor and mature tRNA species are in subpanels I and II, respectively. Exon and intron sequences are represented by filled boxes and horizontal lines, respectively, and probe locations are indicated as lines under the schematics of the pre-tRNA species. Short (IMT1+IMT4) and long (IMT2+IMT3) forms of tRNAiMET are indicated. F–I: Quantification of tRNA precursors (pre-tRNAs) from northern analysis data in panels A–D. Pre-tRNA ratios at each time point were generated by normalising the pre-tRNA species (subpanels I) to the loading control (5S rRNA) and by expressing all the values relative to the 0 h sample of the wild-type strain, which was set to 1.
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pgen-1004716-g002: R-loops at tRNA genes affect pre-tRNA synthesis in strains lacking topoisomerase and RNase H activities.A–E: Wild-type strain BY4741 (WT) and isogenic mutant strains single PGAL-TOP1, triple PGAL-TOP1 rnh1Δ rnh201Δ, double PGAL-TOP1/TOP2 and quadruple PGAL-TOP1/TOP2 rnh1Δ rnh201Δ, were grown at 30°C and harvested at 0 h (galactose- and sucrose- containing medium) and at 6–9 h post-shift to glucose-containing medium. Total RNAs were extracted and analyzed by northern hybridization. The membrane was hybridized separately with probes tRNAiMET (A), tRNA3LEU (B), tRNA2LYS (C) and tRNATRP (D). Ethidium bromide staining of 5S rRNA is in (E). Precursor and mature tRNA species are in subpanels I and II, respectively. Exon and intron sequences are represented by filled boxes and horizontal lines, respectively, and probe locations are indicated as lines under the schematics of the pre-tRNA species. Short (IMT1+IMT4) and long (IMT2+IMT3) forms of tRNAiMET are indicated. F–I: Quantification of tRNA precursors (pre-tRNAs) from northern analysis data in panels A–D. Pre-tRNA ratios at each time point were generated by normalising the pre-tRNA species (subpanels I) to the loading control (5S rRNA) and by expressing all the values relative to the 0 h sample of the wild-type strain, which was set to 1.

Mentions: To assess the outcome of these potentially competing effects, pre-tRNA levels were assessed in strains genetically depleted of Top1 (single PGAL-TOP1), or of both Top1 and Top2 (double PGAL-TOP1 PGAL-TOP2, designated PGAL-TOP1/TOP2 in Fig. 2), or also lacking RNase H activity (triple PGAL-TOP1 rnh1Δ rnh201Δ and quadruple PGAL-TOP1 PGAL-TOP2 rnh1Δ rnh201Δ). Pre-tRNA synthesis was slightly affected under conditions of partial induction of PGAL-TOP1, specifically at 0 h depletion in medium containing galactose plus sucrose (which provides the cells with limited amounts of glucose), in both the triple and quadruple mutant strains, relative to the strains with functional RNase H (Figs. 2A–D panels I, compare lanes 7 and 13 with 1, 4 and 10; quantified in Figs. 2F–I).


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 at tRNA genes affect pre-tRNA synthesis in strains lacking topoisomerase and RNase H activities.A–E: Wild-type strain BY4741 (WT) and isogenic mutant strains single PGAL-TOP1, triple PGAL-TOP1 rnh1Δ rnh201Δ, double PGAL-TOP1/TOP2 and quadruple PGAL-TOP1/TOP2 rnh1Δ rnh201Δ, were grown at 30°C and harvested at 0 h (galactose- and sucrose- containing medium) and at 6–9 h post-shift to glucose-containing medium. Total RNAs were extracted and analyzed by northern hybridization. The membrane was hybridized separately with probes tRNAiMET (A), tRNA3LEU (B), tRNA2LYS (C) and tRNATRP (D). Ethidium bromide staining of 5S rRNA is in (E). Precursor and mature tRNA species are in subpanels I and II, respectively. Exon and intron sequences are represented by filled boxes and horizontal lines, respectively, and probe locations are indicated as lines under the schematics of the pre-tRNA species. Short (IMT1+IMT4) and long (IMT2+IMT3) forms of tRNAiMET are indicated. F–I: Quantification of tRNA precursors (pre-tRNAs) from northern analysis data in panels A–D. Pre-tRNA ratios at each time point were generated by normalising the pre-tRNA species (subpanels I) to the loading control (5S rRNA) and by expressing all the values relative to the 0 h sample of the wild-type strain, which was set to 1.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4214602&req=5

pgen-1004716-g002: R-loops at tRNA genes affect pre-tRNA synthesis in strains lacking topoisomerase and RNase H activities.A–E: Wild-type strain BY4741 (WT) and isogenic mutant strains single PGAL-TOP1, triple PGAL-TOP1 rnh1Δ rnh201Δ, double PGAL-TOP1/TOP2 and quadruple PGAL-TOP1/TOP2 rnh1Δ rnh201Δ, were grown at 30°C and harvested at 0 h (galactose- and sucrose- containing medium) and at 6–9 h post-shift to glucose-containing medium. Total RNAs were extracted and analyzed by northern hybridization. The membrane was hybridized separately with probes tRNAiMET (A), tRNA3LEU (B), tRNA2LYS (C) and tRNATRP (D). Ethidium bromide staining of 5S rRNA is in (E). Precursor and mature tRNA species are in subpanels I and II, respectively. Exon and intron sequences are represented by filled boxes and horizontal lines, respectively, and probe locations are indicated as lines under the schematics of the pre-tRNA species. Short (IMT1+IMT4) and long (IMT2+IMT3) forms of tRNAiMET are indicated. F–I: Quantification of tRNA precursors (pre-tRNAs) from northern analysis data in panels A–D. Pre-tRNA ratios at each time point were generated by normalising the pre-tRNA species (subpanels I) to the loading control (5S rRNA) and by expressing all the values relative to the 0 h sample of the wild-type strain, which was set to 1.
Mentions: To assess the outcome of these potentially competing effects, pre-tRNA levels were assessed in strains genetically depleted of Top1 (single PGAL-TOP1), or of both Top1 and Top2 (double PGAL-TOP1 PGAL-TOP2, designated PGAL-TOP1/TOP2 in Fig. 2), or also lacking RNase H activity (triple PGAL-TOP1 rnh1Δ rnh201Δ and quadruple PGAL-TOP1 PGAL-TOP2 rnh1Δ rnh201Δ). Pre-tRNA synthesis was slightly affected under conditions of partial induction of PGAL-TOP1, specifically at 0 h depletion in medium containing galactose plus sucrose (which provides the cells with limited amounts of glucose), in both the triple and quadruple mutant strains, relative to the strains with functional RNase H (Figs. 2A–D panels I, compare lanes 7 and 13 with 1, 4 and 10; quantified in Figs. 2F–I).

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