GSE122423
The DNA polymerase subunit Pol32 and RNase H1 are required for stress granule formation through R-loop regulation
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. 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.