Limits...
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.

Show MeSH

Related in: MedlinePlus

Cellular RNase H suppresses the mobility of Ty1 LTR-retrotransposons.A: Ty1 elements were analyzed by ChIP-QPCR for distribution of RNA-DNA hybrids in WT (BY4741) and double mutant rnh1Δ rnh201Δ, and in triple mutant PGAL-TOP1 rnh1Δ rnh201Δ and quadruple mutant PGAL-TOP1 rnh1Δ rnh201Δ dbr1Δ depleted of Top1 for 6 h at 30°C. ChIP samples and normalization of Q-PCR values to CEN16 are as in Fig. 1A. The mean of three independent experiments is shown with standard error (two independent experiments for the quadruple mutant). Ab = antibody S9.6. B: S9.6 ChIP-seq profiles over the Ty1 retrotransposon YGRWTY1-1 in the WT (BY4741), double mutant rnh1Δ rnh201Δ, and triple mutant PGAL-TOP1 rnh1Δ rnh201Δ depleted of Top1 for 61h at 30°C. Input chromatin is shown for the WT. Shown below is a graphical representation of a Ty1 element, which is comprised of TYA and TYB open reading frames flanked by long terminal repeats (LTR). The direction of Pol II transcription is indicated by arrowheads. The y-axis represents the relative enrichment of reads where values>1 are above the background level of sequencing (i.e. general intergenic mean, see Materials and Methods). Profiles were generated using the Integrative Genomics Viewer[100]. C: Bar diagrams showing the frequencies of Ty1his3AI mobility after complementation of the wild-type JC3212 (BY4741 TY1his3AI-[Δ1]-3114, [41]) and the mutants double rnh1Δ rnh201Δ and single PGAL-TOP1 with a vector control, and the triple mutant PGAL-TOP1 rnh1Δ rnh201Δ with a vector expressing either wild-type Rnh201 or AGS-related mutant Rnh201G42S[42]. Strains were grown until saturation at 18°C (for growth conditions see Materials and Methods). The frequency of Ty1his3AI mobility is the number of His+ prototrophs divided by the total number of cells plated (see Materials and Methods). The mean of two independent experiments of five independent isolates for each of the strains is shown with standard error. D: PCR analyses showing the integration of Ty1 at the 16 tRNAGLY genes. Upper panel. Graphical representation of the integrated Ty1 element at 5′-flanks of tRNAGLY loci. Primers TYBOUT and SUF16 complementary to Ty1 element and tRNAGLY respectively were used for PCR amplification. Lower panel. Example of SYBR-stained gel showing integration of Ty1 cDNA upstream of any of the 16 tRNAGLY gene loci. Five independent isolates were tested for each strain. Flanking lanes show DNA ladders with lengths in base-pairs (bp). The same yeast cultures served for both analyses in (C) and (D). E: Model for the role of co-transcriptional R-loops in activation of Ty1 retrotransposition (see Discussion). VLP = viral-like particle. Red thick arrow = negative regulation. Green thick arrow = positive regulation. For a detailed review on the mechanisms of TY1 retrotransposition see [36], [37]. See also model in Fig. S7.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4214602&req=5

pgen-1004716-g003: Cellular RNase H suppresses the mobility of Ty1 LTR-retrotransposons.A: Ty1 elements were analyzed by ChIP-QPCR for distribution of RNA-DNA hybrids in WT (BY4741) and double mutant rnh1Δ rnh201Δ, and in triple mutant PGAL-TOP1 rnh1Δ rnh201Δ and quadruple mutant PGAL-TOP1 rnh1Δ rnh201Δ dbr1Δ depleted of Top1 for 6 h at 30°C. ChIP samples and normalization of Q-PCR values to CEN16 are as in Fig. 1A. The mean of three independent experiments is shown with standard error (two independent experiments for the quadruple mutant). Ab = antibody S9.6. B: S9.6 ChIP-seq profiles over the Ty1 retrotransposon YGRWTY1-1 in the WT (BY4741), double mutant rnh1Δ rnh201Δ, and triple mutant PGAL-TOP1 rnh1Δ rnh201Δ depleted of Top1 for 61h at 30°C. Input chromatin is shown for the WT. Shown below is a graphical representation of a Ty1 element, which is comprised of TYA and TYB open reading frames flanked by long terminal repeats (LTR). The direction of Pol II transcription is indicated by arrowheads. The y-axis represents the relative enrichment of reads where values>1 are above the background level of sequencing (i.e. general intergenic mean, see Materials and Methods). Profiles were generated using the Integrative Genomics Viewer[100]. C: Bar diagrams showing the frequencies of Ty1his3AI mobility after complementation of the wild-type JC3212 (BY4741 TY1his3AI-[Δ1]-3114, [41]) and the mutants double rnh1Δ rnh201Δ and single PGAL-TOP1 with a vector control, and the triple mutant PGAL-TOP1 rnh1Δ rnh201Δ with a vector expressing either wild-type Rnh201 or AGS-related mutant Rnh201G42S[42]. Strains were grown until saturation at 18°C (for growth conditions see Materials and Methods). The frequency of Ty1his3AI mobility is the number of His+ prototrophs divided by the total number of cells plated (see Materials and Methods). The mean of two independent experiments of five independent isolates for each of the strains is shown with standard error. D: PCR analyses showing the integration of Ty1 at the 16 tRNAGLY genes. Upper panel. Graphical representation of the integrated Ty1 element at 5′-flanks of tRNAGLY loci. Primers TYBOUT and SUF16 complementary to Ty1 element and tRNAGLY respectively were used for PCR amplification. Lower panel. Example of SYBR-stained gel showing integration of Ty1 cDNA upstream of any of the 16 tRNAGLY gene loci. Five independent isolates were tested for each strain. Flanking lanes show DNA ladders with lengths in base-pairs (bp). The same yeast cultures served for both analyses in (C) and (D). E: Model for the role of co-transcriptional R-loops in activation of Ty1 retrotransposition (see Discussion). VLP = viral-like particle. Red thick arrow = negative regulation. Green thick arrow = positive regulation. For a detailed review on the mechanisms of TY1 retrotransposition see [36], [37]. See also model in Fig. S7.

Mentions: Ty1 LTR-retrotransposons are composed of 2 direct long terminal repeats (LTRs) flanking the TYA and TYB open reading frames (see Fig. 3B; and [36], [37]). TYA encodes the Gag structural proteins of the virus-like particle (VLP), whereas TYB encodes the protease, the integrase and the reverse-transcriptase/RNase H (RT/RNase H). ChIP-QPCR analyses revealed only low levels of RNA-DNA hybrids over Ty1 retrotransposons in the wild-type strain, but notable accumulation was seen in the double mutant rnh1Δ rnh201Δ, and even more in the triple mutant PGAL-TOP1 rnh1Δ rnh201Δ following depletion of Top1 for 6 h (Fig. 3A). S9.6 ChIP-seq profiles showed that RNA-DNA hybrids are unevenly enriched across the Ty1 elements in the RNase H mutants (Figs. 3B and S2). In vitro treatment of S9.6 ChIP samples of double mutant rnh1Δ rnh201Δ with recombinant RNase H strongly reduced the signals over Ty1 retrotransposons confirming thus that these elements are associated with RNA-DNA prone sites (Fig. 1B).


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)

Cellular RNase H suppresses the mobility of Ty1 LTR-retrotransposons.A: Ty1 elements were analyzed by ChIP-QPCR for distribution of RNA-DNA hybrids in WT (BY4741) and double mutant rnh1Δ rnh201Δ, and in triple mutant PGAL-TOP1 rnh1Δ rnh201Δ and quadruple mutant PGAL-TOP1 rnh1Δ rnh201Δ dbr1Δ depleted of Top1 for 6 h at 30°C. ChIP samples and normalization of Q-PCR values to CEN16 are as in Fig. 1A. The mean of three independent experiments is shown with standard error (two independent experiments for the quadruple mutant). Ab = antibody S9.6. B: S9.6 ChIP-seq profiles over the Ty1 retrotransposon YGRWTY1-1 in the WT (BY4741), double mutant rnh1Δ rnh201Δ, and triple mutant PGAL-TOP1 rnh1Δ rnh201Δ depleted of Top1 for 61h at 30°C. Input chromatin is shown for the WT. Shown below is a graphical representation of a Ty1 element, which is comprised of TYA and TYB open reading frames flanked by long terminal repeats (LTR). The direction of Pol II transcription is indicated by arrowheads. The y-axis represents the relative enrichment of reads where values>1 are above the background level of sequencing (i.e. general intergenic mean, see Materials and Methods). Profiles were generated using the Integrative Genomics Viewer[100]. C: Bar diagrams showing the frequencies of Ty1his3AI mobility after complementation of the wild-type JC3212 (BY4741 TY1his3AI-[Δ1]-3114, [41]) and the mutants double rnh1Δ rnh201Δ and single PGAL-TOP1 with a vector control, and the triple mutant PGAL-TOP1 rnh1Δ rnh201Δ with a vector expressing either wild-type Rnh201 or AGS-related mutant Rnh201G42S[42]. Strains were grown until saturation at 18°C (for growth conditions see Materials and Methods). The frequency of Ty1his3AI mobility is the number of His+ prototrophs divided by the total number of cells plated (see Materials and Methods). The mean of two independent experiments of five independent isolates for each of the strains is shown with standard error. D: PCR analyses showing the integration of Ty1 at the 16 tRNAGLY genes. Upper panel. Graphical representation of the integrated Ty1 element at 5′-flanks of tRNAGLY loci. Primers TYBOUT and SUF16 complementary to Ty1 element and tRNAGLY respectively were used for PCR amplification. Lower panel. Example of SYBR-stained gel showing integration of Ty1 cDNA upstream of any of the 16 tRNAGLY gene loci. Five independent isolates were tested for each strain. Flanking lanes show DNA ladders with lengths in base-pairs (bp). The same yeast cultures served for both analyses in (C) and (D). E: Model for the role of co-transcriptional R-loops in activation of Ty1 retrotransposition (see Discussion). VLP = viral-like particle. Red thick arrow = negative regulation. Green thick arrow = positive regulation. For a detailed review on the mechanisms of TY1 retrotransposition see [36], [37]. See also model in Fig. S7.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4214602&req=5

pgen-1004716-g003: Cellular RNase H suppresses the mobility of Ty1 LTR-retrotransposons.A: Ty1 elements were analyzed by ChIP-QPCR for distribution of RNA-DNA hybrids in WT (BY4741) and double mutant rnh1Δ rnh201Δ, and in triple mutant PGAL-TOP1 rnh1Δ rnh201Δ and quadruple mutant PGAL-TOP1 rnh1Δ rnh201Δ dbr1Δ depleted of Top1 for 6 h at 30°C. ChIP samples and normalization of Q-PCR values to CEN16 are as in Fig. 1A. The mean of three independent experiments is shown with standard error (two independent experiments for the quadruple mutant). Ab = antibody S9.6. B: S9.6 ChIP-seq profiles over the Ty1 retrotransposon YGRWTY1-1 in the WT (BY4741), double mutant rnh1Δ rnh201Δ, and triple mutant PGAL-TOP1 rnh1Δ rnh201Δ depleted of Top1 for 61h at 30°C. Input chromatin is shown for the WT. Shown below is a graphical representation of a Ty1 element, which is comprised of TYA and TYB open reading frames flanked by long terminal repeats (LTR). The direction of Pol II transcription is indicated by arrowheads. The y-axis represents the relative enrichment of reads where values>1 are above the background level of sequencing (i.e. general intergenic mean, see Materials and Methods). Profiles were generated using the Integrative Genomics Viewer[100]. C: Bar diagrams showing the frequencies of Ty1his3AI mobility after complementation of the wild-type JC3212 (BY4741 TY1his3AI-[Δ1]-3114, [41]) and the mutants double rnh1Δ rnh201Δ and single PGAL-TOP1 with a vector control, and the triple mutant PGAL-TOP1 rnh1Δ rnh201Δ with a vector expressing either wild-type Rnh201 or AGS-related mutant Rnh201G42S[42]. Strains were grown until saturation at 18°C (for growth conditions see Materials and Methods). The frequency of Ty1his3AI mobility is the number of His+ prototrophs divided by the total number of cells plated (see Materials and Methods). The mean of two independent experiments of five independent isolates for each of the strains is shown with standard error. D: PCR analyses showing the integration of Ty1 at the 16 tRNAGLY genes. Upper panel. Graphical representation of the integrated Ty1 element at 5′-flanks of tRNAGLY loci. Primers TYBOUT and SUF16 complementary to Ty1 element and tRNAGLY respectively were used for PCR amplification. Lower panel. Example of SYBR-stained gel showing integration of Ty1 cDNA upstream of any of the 16 tRNAGLY gene loci. Five independent isolates were tested for each strain. Flanking lanes show DNA ladders with lengths in base-pairs (bp). The same yeast cultures served for both analyses in (C) and (D). E: Model for the role of co-transcriptional R-loops in activation of Ty1 retrotransposition (see Discussion). VLP = viral-like particle. Red thick arrow = negative regulation. Green thick arrow = positive regulation. For a detailed review on the mechanisms of TY1 retrotransposition see [36], [37]. See also model in Fig. S7.
Mentions: Ty1 LTR-retrotransposons are composed of 2 direct long terminal repeats (LTRs) flanking the TYA and TYB open reading frames (see Fig. 3B; and [36], [37]). TYA encodes the Gag structural proteins of the virus-like particle (VLP), whereas TYB encodes the protease, the integrase and the reverse-transcriptase/RNase H (RT/RNase H). ChIP-QPCR analyses revealed only low levels of RNA-DNA hybrids over Ty1 retrotransposons in the wild-type strain, but notable accumulation was seen in the double mutant rnh1Δ rnh201Δ, and even more in the triple mutant PGAL-TOP1 rnh1Δ rnh201Δ following depletion of Top1 for 6 h (Fig. 3A). S9.6 ChIP-seq profiles showed that RNA-DNA hybrids are unevenly enriched across the Ty1 elements in the RNase H mutants (Figs. 3B and S2). In vitro treatment of S9.6 ChIP samples of double mutant rnh1Δ rnh201Δ with recombinant RNase H strongly reduced the signals over Ty1 retrotransposons confirming thus that these elements are associated with RNA-DNA prone sites (Fig. 1B).

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