The Role of Wildtype and Fusion FET Proteins in Transcription Related R-Loop Regulation

Abstract

Co-transcriptional R-loops form when nascent RNA transcripts base pairs with the template DNA strand, displacing the non-template DNA strand. Initially considered a transcriptional byproduct with potential implications for genomic integrity, recent studies highlight the regulatory roles of R-loops. The FET family proteins (FUS, EWSR1, and TAF15) have emerged as significant players in R-loop-mediated transcriptional regulation. This study investigates the mechanisms by which FUS and EWSR1 contribute to transcriptional regulation and global R-loop dynamics. Furthermore, we explore the implications of translocation events leading to the expression of the EWS-FLI1 fusion protein, a hallmark of Ewing Sarcoma present in more than 85% of patients. First, we employed electrophoretic mobility shift assays (EMSA) to show that EWSR1 directly interacts with R-loops through DNA fork regions. The specificity of this interaction is mediated by the RGG1-RRM-RGG2 domains, while the RGG2-ZnF-RGG3 domains contribute to its affinity. Second, we found that both wildtype EWSR1 and the fusion EWS-FLI1 protein interact with RNAPII in large membraneless organelles, where they regulate similar transcripts implicated in Ewing Sarcoma pathogenesis. Next, using in vitro transcription assays, we demonstrate that FUS promotes T7 polymerase-mediated transcription by binding to RNA, thereby inhibiting R- loop formation. This finding is further supported by FUS knockdown in HEK293 cells which leads to the accumulation of R-loops. Lastly, through quantitative mass spectrometry proteomics, we found that aberrant R-loop accumulation disrupts mitochondrial, mitotic, and transport processes in A673 Ewing Sarcoma cells.