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Disorder in the Loop: Identification of a Role for Intrinsic Disorder and Liquid-Liquid Phase Separation in R-Loop Biology
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Summer 2023
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2023-08
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Abstract
R-loops are non-canonical nucleic acid structures composed of a DNA:RNA hybrid, a displaced single-stranded (ss)DNA, and a trailing ssRNA overhang. R-loops perform critical biological functions under normal and disease conditions. To elucidate their cellular functions, we need to understand the mechanisms underlying R-loop formation, recognition, signaling, and resolution. Previous high-throughput screens identified multiple proteins that bind R-loops, including Enzymes and non-enzymatic Readers. However, the precise mechanisms by which these proteins modulate R-loop functions are not fully known. The Fragile X Protein (FMRP) has been recently shown to prevent R-loop-mediated DNA double strand breaks (DSBs), but the mechanism was unknown. FMRP has been previously shown to undergo Liquid-Liquid Phase Separation (LLPS) by itself and with another non-canonical nucleic acid structure, RNA G-quadruplexes, via its C-terminal intrinsically disordered region (C-IDR). Here, we identified the same C-IDR as the predominant R-loop binding site. This unexpected discovery prompted us to explore the hypothesis that disordered regions of other R-loop binding proteins are also utilized to recognize R-loops. Our analysis of the R-loop interactome revealed that low-complexity IDRs are prevalent in this interactome, and that Readers and Enzyme IDRs are distinct (Gly, Ser, Arg, and Pro-rich vs. Glu, Lys, Arg, and Ser-rich, respectively). Furthermore, like FMRP, both R-loop Readers and Enzymes are not only modular, (i.e., contain folded domain(s) interspersed with IDRs), but are also predicted to undergo LLPS. Next, we demonstrated that the IDRs from the R-loop binding protein RBM3 and the R-loop helicase DDX21 also bind to R-loops, providing additional examples of IDR-mediated R-loop binding from an R-loop Reader and Enzyme, respectively. Finally, we demonstrated that FMRP C-IDR and DDX21 N-IDR can undergo co-LLPS with R-loops suggesting that IDR-based R-loop binding and co-LLPS is a universal mechanism shared by all members of the interactome. Therefore, we propose that IDRs can provide a functional link between R-loop recognition and downstream signaling through the assembly of LLPS-mediated membrane-less R-loop foci, where the activities of the folded domains are coordinated to regulate the biological functions of R-loops. Mutations or dysregulation of the function of IDR-enriched R-loop interactors can potentially lead to severe genomic defects, such as the R-loop-mediated DSBs observed in Fragile X patient-derived cells.