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

ABSTRACT: 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.

ORGANISM(S): Schizosaccharomyces pombe

PROVIDER: GSE101086 | GEO | 2018/06/01

REPOSITORIES: GEO

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Project description:Strong cellular stress causes wide-spread perturbations in the transcriptome and in several types of DNA/RNA interactions, and through incompletely understood pathways to formation of cytoplasmic stress granules (SGs). We have investigated the relationships between strong transcriptional induction, RNA:DNA hybrid stretches (R-loops), and SG formation under severe hyperosmotic and glucose stress. Several mutations affecting DNA processing proteins, including the DNA polymerase subunit Pol32, confer SG formation defects. Severe stress increased R loop levels globally. We found that facilitating removal of R-loops by RNase H overexpression, with activity towards RNA:DNA hybrids, accelerated and enhanced transcriptional induction of stress-activated genes. Thus, overexpression of RNase H1, but not RNase H2, reduced R-loops globally around the 1 h mark. Remarkably, it also reduced SG formation. We performed a genome-wide analysis of the induction or repression kinetics of gene expression under severe stress conditions. RNase H1 overexpression reduced R-loops locally in highly transcribed stress-affected genes, as expected. Notably, it also increased expression of several stress-induced genes. Conversely, in cells where R-loops are not efficiently resolved, transcriptional induction of the same genes under stress was muted and occurred with a delay. Thus, in pol32∆ mutants, where SG accumulation is delayed, R-loop levels are elevated, and induction of stress genes suppressed. The pol32∆ mutants are also refractory to the effects of RNase H1 overexpression on stress gene induction, R-loop resolution, and SG formation, indicating that Pol32 may act downstream of Rnh1 in the regulation of these three processes. These findings demonstrate an unexpected link between R-loops and formation of SGs. Together, these observations indicate that under stress, strong transcriptional induction of specific genes or genomic regions causes R-loop accumulation, which then requires RNase H1 activity for resolution. If unresolved, the accumulated R-loops impede continued stress-induced transcription, and delay or prevent SG formation.

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

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