Binding of the nuclear ribonucleoprotein family member FUS to RNA prevents R-loop RNA:DNA hybrid structures.

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. Here, 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. The 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 from 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 the potential to affect transcription by any RNA polymerase, highlighting the broad impact FUS can have on RNA metabolism in cells and disease.

Fusion protein EWS-FLI1 is incorporated into a protein granule in cells.

Ewing sarcoma is driven by fusion proteins containing a low complexity (LC) domain that is intrinsically disordered and a powerful transcriptional regulator. The most common fusion protein found in Ewing sarcoma, EWS-FLI1, takes its LC domain from the RNA-binding protein EWSR1 (Ewing Sarcoma RNA-binding protein 1) and a DNA-binding domain from the transcription factor FLI1 (Friend Leukemia Virus Integration 1). EWS-FLI1 can bind RNA polymerase II (RNA Pol II) and self-assemble through its low-complexity (LC) domain. The ability of RNA-binding proteins like EWSR1 to self-assemble or phase separate in cells has raised questions about the contribution of this process to EWS-FLI1 activity. We examined EWSR1 and EWS-FLI1 activity in Ewing sarcoma cells by siRNA-mediated knockdown and RNA-seq analysis. More transcripts were affected by the EWSR1 knockdown than expected and these included many EWS-FLI1 regulated genes. We reevaluated physical interactions between EWS-FLI1, EWSR1, and RNA Pol II, and employed a cross-linking based strategy to investigate protein assemblies associated with the proteins. The LC domain of EWS-FLI1 was required for the assemblies observed to form in cells. These results offer new insights into a protein assembly that may enable EWS-FLI1 to bind its wide network of protein partners and contribute to regulation of gene expression in Ewing sarcoma.