Towards understanding R-loop regulation by RNAse H1 in saccharomyces cerevisiae

Abstract

An RNA:DNA hybrid refers to the base pairing of RNA and DNA. In particular, R-loops are three-stranded nucleic acid structures composed of an RNA:DNA hybrid and a displaced DNA strand. In the recent years, several studies have shown that R-loops impact multiple biological processes, such as transcription, telomere maintenance, and DNA repair. Indeed, failure to resolve R-loops in a timely manner results in genomic instability. Hence, cells have evolved several mechanisms to tightly regulate R-loops. One of the most prominent example of an RNA:DNA hybrid removal factor is RNase H1, a monomeric enzyme that degrades the RNA moiety of RNA:DNA hybrids. In human cells, Replication Protein A (RPA) appears to be a regulator of RNase H1 by promoting its recruitment and activity on R-loops. However, it remains unclear if RPA or other proteins influence RNase H1 activity in yeast. RNase H1 is recruited to the chromatin in conditions with high R-loop levels, suggesting that RNase H1 binds to accumulated R-loops. However, how RNase H1 is regulated and responds to high R-loop levels is still elusive. In this study, we have employed in vitro and in vivo experiments to understand where, when and how RNase H1 binds to R-loops. We confirm that RNase H1 is recruited to loci with accumulated R-loops, especially in RNAPIII-transcribed genes. Furthermore, we show that RNase H1 can remove R-loops in all cell cycle phases, allowing RNase H1 to maintain R-loop homeostasis in all conditions, including outside of a replication-transcription conflict context. Using co-immunoprecipitation experiments, we confirm that RPA interacts with the first Hybrid-binding (HB) domain of RNase H1 in a DNA-dependent manner. However, overexpression of RNase H1 without the first HB domain is able to remove R-loops in vivo, suggesting that RNase H1 overexpression does not require RPA interaction to remove R-loops in yeast. Moreover, using in vivo and in vitro assays, we show that the second HB domain of RNase H1 is sufficient to promote binding and removal of R-loops, unlike what was described. We confirm that both HB domains of RNase H1 contribute to a stable interaction with R-loops and likely both HB domains have different functions in RNase H1 binding to substrates. Taken together, these results shed light on how RNase H1 recognizes and responds to stable RNA-DNA hybrids.