Regulation of human RNase H1 by phosphorylation
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Abstract
R-loops, structures consisting of an RNA:DNA hybrid and a displaced DNA strand, are increasingly investigated as a cell-intrinsic and cancer-relevant source of replication stress (RS). On the other hand, RS stabilizes R-loops in actively replicating and transcribing regions. This suggests potential unexplored mechanisms through which cells mitigate toxic accumulation of RS-borne R-loops.
To investigate the relationship between the replication stress and R-loops, we first stratified RS by severity in HCT116 cells, reasoning that mild RS might have a distinct signaling signature in comparison to moderate or strong RS. Next, we employed multiplexed mass spectrometry-based phosphoproteomics to investigate the regulation of R-loop resolving enzymes upon induction of different levels of RS. Comparisons of aphidicolin- and oncogene-induced RS revealed similarities at the phosphoproteome level, and a common loss of phosphorylation on RNA processing factors. Interestingly, we found that RS reduces phosphorylation of a major R-loop nuclease, RNase H1, at three residues (S74, S86 and S80) adjacent to its hybrid-binding domain.
The kinase search revealed that DYRK1A phosphorylates RNase H1 at S74. Phosphoproteomics analyses revealed that the reduction in DYRK1A signaling is a driver of RS-mediated loss of phosphorylation on RNA processing factors. Finally, we explored functions of RNase H1 phosphorylation. In in vitro assays, the triple phospho-mimicking (S3xD) mutant of RNase H1 had decreased binding to RNA:DNA hybrids in comparison to wild-type or phospho-dead (S3xA) mutant, implying that phosphorylation directly impacts binding to hybrids. In cells, the S3xD mutant showed decreased separation between nucleoli and the nucleoplasm in comparison to S3xA. Finally, interactome analyses with the phospho-mutants and after DYRK1A inhibition revealed phosphorylation-based interactors of RNase H1 and implicated GSK3β in the regulation of RNase H1 stability.
In summary, this work presents major advancements in the understanding of DYRK1A signaling, and of the regulation of activity and stability of RNase H1 by phosphorylation. This could impact the use of RNase H1 as a tool in R-loop biology and advance the understanding of DYRK1A function in disease.