Investigating the mechanism of interaction of R-loops and the Fragile X protein, FMRP: an entanglement of disordered tails and multivalency

Abstract

Fragile X syndrome (FXS) is one of the most prevalent forms of inherited intellectual disability and is the leading monogenetic cause of autism spectral disorder. FXS is caused by lack of expression or mutations of the FMR1 gene which encodes fragile X messenger ribonucleoprotein, FMRP. Recently, FMRP has been shown to undergo liquid-liquid phase separation (LLPS) in vitro and to localize different isoforms in distinct membrane-less organelles in cellulo. Despite three decades of research, the molecular mechanisms by which FMRP functions are still not fully understood. FMRP is best known as a cytoplasmic mRNA-binding translational regulator. Although the presence of a small fraction of FMRP in the nucleus has long been realized, it was only recently that studies are beginning to uncover its role in influencing genomic function and stability [1]. The Feng lab recently discovered a novel genome protective role for FMRP. FXS patient-derived cells undergo higher level of DNA double-strand breaks (DSBs) than normal cells, especially during DNA replication stress. These DSBs occur at sequences prone to forming R-loops, which are co-transcriptional RNA:DNA hybrids associated with multiple functions including genome instability. Exogenously expressed WT FMRP, but not an I304N disease-causing mutant abates R-loop-induced DSBs. This unexpected finding suggests that FMRP promotes genome integrity by preventing R-loop accumulation and chromosome breakage. However, the mechanism by which FMRP performs this critical function, and how disease-causing mutations affect this process is not fully understood. Here, we set out to elucidate the mechanism underlying FMRP's role in maintaining genome stability. First, we demonstrate that FMRP directly binds R-loops primarily through its C-terminal Intrinsically Disordered Region (C-IDR). In FMR1 CRISPR knock-out HEK293T cells, we observed dynamic condensates in WT FMRP but not in I304N mutant, suggesting that this mutation, located in the central RNA binding KH2 domain, disrupted the ability of I304N to assemble into higher order condensates. Furthermore, unlike the I304N FMRP, WT FMRP show increase in nuclear condensates that overlap with R-loops under replication stress. While we found that WT and I304N mutant can co-phase separate with R-loops in vitro, WT FMRP tends to form hollow droplets with R-loop substrates localized at the periphery, but the vast majority of I304N droplets are filled with dispersed R-loops substrates. Taken together, these data support the hypothesis that the ability of FMRP to form higher order assemblies with R-loops is critical to maintaining genome stability. Our study sets the stage to test the proposed phase separation-function paradigm in other FXS disease mutants.