Lay, M. A. | Search bioRxiv

bioRxiv2024

Ewing Sarcoma Related protein 1 recognizes R-loops by binding DNA forks

EWSR1 (Ewing Sarcoma Related protein 1) is an RNA binding protein that is ubiquitously expressed across cell lines and involved in multiple parts of RNA processing, such as transcription, splicing, and mRNA transport. EWSR1 has also been implicated in cellular mechanisms to control formation of R-loops, a three-stranded nucleic acid structure consisting of a DNA:RNA hybrid and a displaced single-stranded DNA strand. Unscheduled R-loops result in genomic and transcription stress. Loss of function of EWSR1 functions commonly found in Ewing Sarcoma correlates with high abundance of R-loops. In this study, we investigated the mechanism for EWSR1 to recognize an R-loop structure specifically. Using electrophoretic mobility shift assays (EMSA), we detected the high affinity binding of EWSR1 to substrates representing components found in R-loops. EWSR1 specificity could be isolated to the DNA fork region, which transitions between double- and single-stranded DNA. Our data suggests that the Zinc-finger domain (ZnF) with flanking arginine and glycine rich (RGG) domains provide high affinity binding, while the RNA recognition motif (RRM) with its RGG domains offer improved specificity. This model offers a rational for EWSR1 specificity to encompass a wide range in contexts due to the DNA forks always found with R-loops.

biochemistry↗

bioRxiv2022

A role for phase separation to prevent R-loops during transcription

The protein FUS (FUSed in sarcoma) is a metazoan RNA-binding protein that influences RNA production by all three nuclear polymerases. FUS also binds nascent transcripts, RNA processing factors, RNA polymerases, and transcription machinery. We explored the role of FUS binding interactions for activity during transcription. In vitro run-off transcription assays revealed FUS enhanced RNA produced by a non-eukaryote polymerase. Activity also reduced the formation of R-loops between RNA products and their DNA template. Analysis by domain mutation and deletion indicated RNA-binding was required for activity. We interpret that FUS binds and sequesters nascent transcripts to prevent R-loops forming with nearby DNA. DRIP-seq analysis showed that a knockdown of FUS increased R-loop enrichment near expressed genes. Prevention of R-loops by FUS binding to nascent transcripts has potential to affect transcription by any RNA polymerase, highlighting the broad impact FUS can have on RNA metabolism in cells and disease.

biochemistry↗