RNA Processing Factors Swd2.2 and Sen1 Antagonize RNA Pol III-Dependent Transcription and the Localization of Condensin at Pol III Genes

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The Affinity of the S9.6 Antibody for Double-Stranded RNAs Impacts the Accurate Mapping of R-Loops in Fission Yeast

R-loops, which result from the formation of stable DNA:RNA hybrids, can both threaten genome integrity and act as physiological regulators of gene expression and chromatin patterning. To characterize R-loops in fission yeast, we used the S9.6 antibody-based DRIPc-seq method to sequence the RNA strand of R-loops and obtain strand-specific R-loop maps at near nucleotide resolution. Surprisingly, preliminary DRIPc-seq experiments identified mostly RNase H-resistant but exosome-sensitive RNAs that mapped to both DNA strands and resembled RNA:RNA hybrids (dsRNAs), suggesting that dsRNAs form widely in fission yeast. We confirmed in vitro that S9.6 can immuno-precipitate dsRNAs and provide evidence that dsRNAs can interfere with its binding to R-loops. dsRNA elimination by RNase III treatment prior to DRIPc-seq allowed the genome-wide and strand-specific identification of genuine R-loops that responded in vivo to RNase H levels and displayed classical features associated with R-loop formation. We also found that most transcripts whose levels were altered by in vivo manipulation of RNase H levels did not form detectable R-loops, suggesting that prolonged manipulation of R-loop levels could indirectly alter the transcriptome. We discuss the implications of our work in the design of experimental strategies to probe R-loop functions

The Affinity of the S9.6 Antibody for Double-Stranded RNAs Impacts the Accurate Mapping of R-Loops in Fission Yeast

R-loops, which result from the formation of stable DNA:RNA hybrids, can both threaten genome integrity and act as physiological regulators of gene expression and chromatin patterning. To characterize R-loops in fission yeast, we used the S9.6 antibody-based DRIPc-seq method to sequence the RNA strand of R-loops and obtain strand-specific R-loop maps at near nucleotide resolution. Surprisingly, preliminary DRIPc-seq experiments identified mostly RNase H-resistant but exosome-sensitive RNAs that mapped to both DNA strands and resembled RNA:RNA hybrids (dsRNAs), suggesting that dsRNAs form widely in fission yeast. We confirmed in vitro that S9.6 can immuno-precipitate dsRNAs and provide evidence that dsRNAs can interfere with its binding to R-loops. dsRNA elimination by RNase III treatment prior to DRIPc-seq allowed the genome-wide and strand-specific identification of genuine R-loops that responded in vivo to RNase H levels and displayed classical features associated with R-loop formation. We also found that most transcripts whose levels were altered by in vivo manipulation of RNase H levels did not form detectable R-loops, suggesting that prolonged manipulation of R-loop levels could indirectly alter the transcriptome. We discuss the implications of our work in the design of experimental strategies to probe R-loop functions

Lack of Top1 further increases the association of condensin with Pol III-transcribed genes when Swd2.2 and Sen1 are missing.

<p><b>A</b>. Serial dilutions of the indicated strains were plated on rich media at the indicated temperatures. <b>B</b>. Chromosome segregation in anaphase was monitored in the indicated strains after growing cells for one generation at 34°C. For each genotype, a minimum of 6 independent experiments was performed in which a minimum of 100 anaphase cells were scored (***<0.001; **<0.01 Wilcoxon - Mann Whitney). Anaphases were scored as defective when chromatin was detected lagging between the two main DNA masses <b>C</b>. ChIP qPCR of the indicated strains grown in cycling conditions at the indicated loci (mean ± standard deviation from 6 biological replicates. NS: not significant *P<0.05; **P<0.01; ***P<0.001 Wilcoxon - Mann Whitney). <b>D</b>. Model. Lack of Swd2.2 and Sen1 increases gene transcription at Pol III-transcribed genes. According to the twin supercoiled domain model, this results in more positive supercoils downstream of the polymerase and compensatory negative supercoils upstream of the polymerase. Negative supercoils favor the formation of R-Loops (reviewed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004794#pgen.1004794-Drolet1" target="_blank">[41]</a>). Positive supercoils result in nucleosome eviction. This topological stress also facilitates the recruitment of condensin, either directly or indirectly.</p

Transcription is enhanced at Pol III-transcribed genes when Swd2.2 and Sen1 are missing.

<p><b>A</b>. Sen1 is enriched at Pol III-transcribed genes. ChIP qPCR of the indicated strains grown in cycling conditions at the indicated loci (mean ± standard deviation from 3 biological replicates). NTS#2 is a site within the Replication Fork Barrier of the rDNA and is shown as a comparison. <b>B</b>. Flag-tagged Sen1 co-immunoprecipitates with Myc-tagged Rpc25. Whole cell extracts (WCE) and the immuno-precipitated material (Flag IP) of the indicated strains were analyzed by western blot. <b>C</b>. Rpc25 becomes more abundant at Pol III-transcribed genes when Swd2.2 and Sen1 are missing. ChIP qPCR of the indicated strains grown in cycling conditions at the indicated loci (mean ± standard deviation from 3 biological replicates). <b>D</b>. Western blot analysis of the stability of Rpc25-13myc. Tubulin is used as a loading control. <b>E</b>. Pol III transcripts are more abundant when Swd2.2 and Sen1 are missing. Total RNAs extracted from swd2.2+sen1+ or <i>swd2.2Δsen1Δ</i> cells grown in rich medium at 30°C were analyzed by RT-qPCR (3 biological replicates, 2 RT per replicate).</p

The double deletion of Swd2.2 and Sen1 facilitates the localization of condensin at Pol III-transcribed genes.

<p><b>A</b>. Serial dilutions of the indicated strains were plated on rich media at the indicated temperatures. <b>B</b>. Chromosome segregation in anaphase was monitored in the indicated strains after growing cells for one generation at 34°C. For each genotype, a minimum of 6 independent experiments was performed in which a minimum of 100 anaphase cells were scored (***<0.001; **<0.01 Wilcoxon - Mann Whitney). Anaphases were scored as defective when chromatin was detected lagging between the two main DNA masses <b>C</b>. ChIP-qPCR analysis of the amount of GFP-tagged Cut3 cross-linked to chromatin in cell populations of the indicated genotypes grown at 30°C (mean ± standard deviation from 6 biological replicates (NS: not significant, *P<0.05; **P<0.01; ***P<0.001 Wilcoxon - Mann Whitney). The primers used in this study are shown on <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004794#pgen.1004794.s013" target="_blank">Table S1</a>. <b>D</b>. Mitotic indexes of the cell populations used in <b>C</b>. Cells were fixed with cold methanol and processed for immuno-fluorescence using an anti-tubulin antibody. Cells with a spindle were counted as mitotic.</p

Lack of Swd2.2 and Sen1 results in local topological stress at Pol III-transcribed genes.

<p><b>A</b>. ChIP qPCR of the indicated strains grown in cycling conditions at the indicated loci (mean ± standard deviation from 6 biological replicates. NS: not significant *P<0.05; **P<0.01; ***P<0.001 Wilcoxon - Mann Whitney). <b>B</b>. Western blot analysis of the stability of Top1-3flag. Tubulin is used as a loading control. <b>C</b>. ChIP qPCR of the indicated strains grown in cycling conditions at the indicated loci (mean ± standard deviation from 6 biological replicates. NS: not significant *P<0.05; **P<0.01; ***P<0.001 Wilcoxon - Mann Whitney). <b>D</b>. Western blot analysis of the stability of Top2-GFP. Tubulin is used as a loading control. <b>E</b>. ChIP qPCR of histone H3 in the indicated strains grown in cycling conditions at the indicated loci (mean ± standard deviation from 6 biological replicates. NS: not significant *P<0.05; **P<0.01; Wilcoxon - Mann Whitney).</p

R-Loops form in abundance at Pol III-transcribed genes but they do not significantly impact the association of condensin.

<p><b>A</b>. ChIP qPCR of the indicated strains grown in cycling conditions at the indicated loci (mean ± standard deviation from 3 biological replicates). <b>B</b>. As in <b>A</b>. Cells were grown in minimal medium for a minimum of 18 hours to promote the over-expression of RnhA driven by the nmt promoter. <b>C</b>. Genomic DNA was extracted from rnh1+rnh201+ and <i>rnh1Δrnh201Δ</i> cells in preparation for the DRIP procedure. Equal amount of genomic DNA were spotted on a nylon membrane and incubated with 2 µg/mL of purified S9.6 antibody. The amount of S9.6 bound to the DNA was revealed using chemiluminescence. <b>D</b>. DRIP-qPCR of the indicated strains grown in cycling conditions at the indicated loci (mean ± standard deviation from 3 biological replicates). <b>E</b>. Cells of the indicated genotypes were grown in minimum medium lacking thiamine for a minimum of 18 hours to drive the over-expression of RnhA. ChIP-qPCR was then performed (mean ± standard deviation from 3 biological replicates).</p

Senataxin homologue Sen1 is required for efficient termination of RNA polymerase III transcription

International audienceR‐loop disassembly by the human helicase Senataxin contributes to genome integrity and to proper transcription termination at a subset of RNA polymerase II genes. Whether Senataxin also contributes to transcription termination at other classes of genes has remained unclear. Here, we show that Sen1, one of two fission yeast homologues of Senataxin, promotes efficient termination of RNA polymerase III (RNAP3) transcription in vivo. In the absence of Sen1, RNAP3 accumulates downstream of RNAP3‐transcribed genes and produces long exosome‐sensitive 3′‐extended transcripts. Importantly, neither of these defects was affected by the removal of R‐loops. The finding that Sen1 acts as an ancillary factor for RNAP3 transcription termination in vivo challenges the pre‐existing view that RNAP3 terminates transcription autonomously. We propose that Sen1 is a cofactor for transcription termination that has been co‐opted by different RNA polymerases in the course of evolution

The extruded non-template strand determines the architecture of R-loops

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