A role for phase separation to prevent RNA:DNA hybrids

The function of phase separation for many RNA-binding proteins is poorly understood, including FUS (FUSed in sarcoma). FUS binds RNA Pol II and controls phosphorylation of serine-2 (Ser2P). We uncovered a FUS activity involving no interactions with the polymerase. FUS could activate transcription of a non-eukaryote polymerase by preventing RNA invasion of DNA to form R-loops. This activity required binding to the nascent transcript and phase separation. We confirmed the activity in human cells using DRIP-seq, where R-loop sites formed were consistent with characteristic features of FUS activity, including transcription start sites where Ser2P signals accumulate after FUS knockdown. R-loop sites were enriched for sequences favored by the degenerate binding specificity of FUS. FUS also regulated R-loops near active retrotransposons and regions associated with DNA repeat expansion disorders. These findings offer a unified model that incorporates RNA-binding and phase separation to guard against instability from disruptive nucleic acid structures. Overall design: Comparison of R-loops using DRIP-seq with and without knockdown of FUS by siRNA