Droplets, tails and loops: The role of intrinsic disorder in R-loop biology.

Abstract

R-loops are non-canonical triple-stranded RNA:DNA hybrid structures that perform critical biological functions. However, dysregulated R-loops can also cause cancer and neurological disorders. Previous high-throughput studies have identified over a thousand proteins involved in recognizing, preventing and resolving R-loops, with many of them containing both folded nucleic acid-binding/processing domains and Intrinsically Disordered Regions (IDRs). Nonetheless, the precise molecular mechanisms by which these proteins regulate R-loop function remain elusive. While investigating the Fragile-X Syndrome Protein (FMRP), we unexpectedly discovered that the C-Terminal IDR of FMRP, which promotes Liquid-Liquid Phase Separation (LLPS) of FMRP, is the predominant R-loop binding site, with the N-terminal domains recognizing the trailing ssRNA overhang. In addition, we have recently shown that FMRP can suppress persistent R-loops that form during transcription, a process that is also enhanced by LLPS via the assembly of membraneless transcription factories. These exciting findings prompted us to undertake a combined in-cellulo/in-silico/in-vitro approach to investigate the mechanisms of R-loop recognition, signaling, and resolution. We observed that, like FMRP, the majority of proteins in the R-loop interactome contains long IDRs that are highly enriched in low complexity sequences with biased amino acid composition. Furthermore, our analysis shows that several proteins in the R-loop interactome are either predicted to or have been experimentally demonstrated to undergo LLPS or are known to be associated with phase separated membraneless organelles. Finally, we have recently demonstrated in vitro that the IDRs of different proteins selected from the R-loop interactome can also directly recognize R-loops and undergo LLPS. Our overall results present a thought-provoking hypothesis that IDRs in the R-loop interactome provide a functional link between R-loop recognition via direct binding and downstream signaling through the assembly of LLPS-mediated membraneless R-loop foci.