FUS modulates R-loops by functionally interacting with RNase H1.

R-loops are three-stranded nucleic acid structures consisting of an RNA:DNA hybrid and a displaced single-stranded DNA, typically formed during transcription. Emerging evidence indicates that R-loops are not merely transcriptional byproducts, but serve as functional regulatory structures that influence chromatin organization, transcriptional pausing, and RNA processing. However, dysregulated accumulation of R-loops can induce DNA damage and genomic instability, necessitating precise mechanisms for their regulation. This study aims to elucidate the role of the RNA-binding protein FUS (Fused in Sarcoma), a protein mutated in Amyotrophic Lateral Sclerosis (ALS) and cancer, in modulating R-loop dynamics. Knockdown of FUS in HeLa cells resulted in a significant increase in global R-loop levels, as assessed by immunofluorescence and dot blot assays. Proximity ligation assay (PLA) demonstrated that FUS is in close proximity to R-loops and nascent RNA. Further, FUS was found to interact with RNase H1, a key endonuclease involved in R-loop resolution, in an R-loop dependent manner, as demonstrated by PLA and co-immunoprecipitation assay. Importantly, in vitro assays show that FUS enhances RNase H1-mediated degradation of RNA:DNA hybrids. Moreover, FUS depletion reduces RNase H1 proximity to elongating RNA polymerase II, suggesting altered engagement of RNase H1 with the transcription machinery. These findings highlight a crucial role for FUS-RNase H1 axis in regulating R-loop levels, providing insights into the potential mechanisms underlying R-loop-associated pathologies in neurodegenerative diseases linked to FUS.