ENAapplication/xmlftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR818/005/SRR8180995/SRR8180995.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR818/006/SRR8180996/SRR8180996.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR818/004/SRR8180994/SRR8180994.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR818/002/SRR8181002/SRR8181002.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR818/009/SRR8180999/SRR8180999.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR818/007/SRR8180997/SRR8180997.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR818/001/SRR8181001/SRR8181001.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR818/008/SRR8180998/SRR8180998.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR818/000/SRR8181000/SRR8181000.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR818/003/SRR8180993/SRR8180993.fastq.gzprimary200OKGenomicsChemistry and Molecular Biology, University of Gothenburghttps://www.ebi.ac.uk/ena/browser/view/PRJNA505169Saccharomyces cerevisiaeStrong 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. Overall design: Samples were collected every 30 min after onset of 1.5 M KCl stress, up to 2 h. Samples were collected from two independent replicate experiment time series Total number of samples = 10. Control samples were collected at the 0 h time point.ENAfalseThe DNA polymerase subunit Pol32 and RNase H1 are required for stress granule formation through R-loop regulationThe DNA polymerase subunit Pol32 and RNase H1 are required for stress granule formation through R-loop regulation2025-09-242022-01-01PRJNA505169GSE1224234932