Talk Abstracts

Talk on Friday 03:45-04:00pm submitted by Victor Corral

RNase H1 phosphorylation promotes RPA interaction to safeguard genome integrity from R-loop-associated instability

Victor M. Corral (Molecular Pharmacology and Therapeutics Graduate Program, Department of Pharmacology, University of Minnesota, Minneapolis MN), Wannasiri Chiraphapphaiboon (The Masonic Cancer Center, University of Minnesota, Minneapolis MN), Niraja A. Soman (Molecular Pharmacology and Therapeutics Graduate Program, Department of Pharmacology, University of Minnesota, Minneapolis MN), Hai Dang Nguyen (Department of Pharmacology; and The Masonic Cancer Center, University of Minnesota, Minneapolis MN)

Abstract:
Genomic instability arises from a variety of cellular processes, including DNA replication and transcription. R loops are transcription intermediates resulting from the formation of stable RNA:DNA hybrids and a displaced single-stranded DNA (ssDNA). Although R loops have important physiological roles under normal conditions, aberrant accumulation and/or distribution of R loops in the genome become a source of genomic instability and are associated with cancers. R loops must be tightly regulated to prevent R-loop-associated genomic instability. RNase H1 is an enzyme that degrades the RNA moiety within RNA:DNA hybrids of R loops. We previously showed that Replication Protein A (RPA), a ssDNA-binding protein that coats the displaced ssDNA at R loops, is a sensor of R loops and interacts with RNase H1 to promote R-loop resolution⁽¹⁾. How RPA:RNase H1 interaction is regulated is not known. Here, we identified RNase H1 is phosphorylated at Ser76 and Ser233 in human cells. Interestingly, Ser233 phosphorylation is independent of Ser76 phosphorylation, suggesting potentially two independent phosphorylation signaling cascades. Since RNase H1^S76 residue is adjacent to its RPA interacting motif, we hypothesized that RNase H1^S76 phosphorylation plays a role in resolving R loops through its interaction with RPA. We generated a phospho-specific antibody and detected endogenous RNase H1 phosphorylation at Ser76. Moreover, phosphorylated RNase H1^S76 (pRNase H1^S76) is associated with R loops and depends on its ability to bind to R loops, but not RPA interaction. Conversely, RNase H1^S76A phospho-mutant disrupted RPA interaction to a similar extent as the previously reported RPA-defective binding mutant RNase H1^R57A. As a consequence, the RNase H1^S76A phospho-mutant failed to suppress PARP inhibitor-induced R-loops and R-loop-associated genomic instability. Lastly, pharmacologic inhibition of DNA checkpoint kinases, ATM/ATR, or cyclin-dependent kinases, CDKs, did not suppress pRNase H1^S76, suggesting an unknown kinase involved in R-loop regulation. Our results demonstrate a mechanistic detail in RPA:RNase H1 interaction mediated by RNase H1 phosphorylation at Ser76 to promote R-loop resolution.

References:
1) Nguyen, H. D., et al. (2017). Molecular Cell.

Keywords: R loops, RNase H1, Phosphorylation