Improved mapping and characterization of R-loop forming loci in fission yeast.

R-loops both threaten genome integrity and act as physiological regulators of gene expression and chromatin patterning. To characterize R-loop forming loci in the fission yeast, we used the S9.6-based DRIPc-seq approach to obtain improved strand-specific maps of R-loop forming loci at near nucleotide resolution. We show that the weak affinity of the S9.6 antibody for double-stranded RNA (dsRNA) is sufficient to confound the mapping of genuine R-loops by this approach. dsRNA elimination allowed the identification of two distinct classes of R-loops that differ by their sensitivity to endogenous RNase H activity. Both classes associate with common chromatin features, which are similar to those associated with R-loop formation in human. We used RNA-seq to identify transcripts whose steady-state levels were affected by genome-wide manipulation of R-loop levels. gdh2 is such a transcript and we show that extra RNase H1 stimulates the cis-acting transcription interference between an upstream non-coding RNA and gdh2. Surprisingly, we found that most transcripts whose levels were altered by in vivo manipulation of R-loop levels did not form R-loops. Conversely, the abundance of only few R-loop forming transcripts was impacted by genome-wide variation of R-loop levels. We conclude that prolonged manipulation of R-loop levels imparts indirect effects on the transcriptome that could complicate the use of this strategy to understand R-loop functions. DNA-RNA immunoprecipitation strand-specific (DRIPc-seq) were performed on Schizosaccaromyces pombe cells from different genotypes (Wild-type, RNaseH1 overexpressed, RRP6 KO, RNaseH1+2 KO). Genomic DNA was treated with either RNaseA, RNaseA+H, RNaseA+III prior DRIPc-seq. Total RNA-seq was performed on Schizosaccaromyces pombe cells treated with several conditions (Wild-type, RNaseH1 overexpression, RNaseH1+2 KO). Small RNA-seq was performed on Schizosaccaromyces pombe Wild-type cells.