Promoter R-loops Recruit U2AF1 to Modulate Its Phase Separation and RNA Splicing
R-loops and guanine quadruplexes (G4s) are secondary structures of nucleic acids that are ubiquitously present in cells, and are enriched in promoter regions of genes. By employing a bioinformatic approach based on overlap analysis of transcription factor ChIP-seq datasets, we found that many splicing factors, including U2AF1 whose recognition of 3¢ splicing site is crucial for pre-mRNA splicing, exhibit pronounced enrichment at endogenous R-loop- and DNA G4-structure loci in promoter regions of human genes. We revealed that U2AF1 can bind directly to R-loops and DNA G4 structures at low nM binding affinity. Additionally, we showed the ability of U2AF1 to undergo phase separation, which could be stimulated by binding with R-loops, but not duplex DNA, RNA/DNA hybrid, DNA G4, or single-stranded RNA. We also demonstrated that U2AF1 binds to promoter R-loops in human cells, and this binding competes with U2AF1's interaction with 3¢-splicing site, thereby modulating pre-mRNA splicing. Together, we uncovered a group of candidate proteins that can bind to both R-loops and DNA G4s, revealed the direct and strong interaction of U2AF1 with these nucleic acid structures, and unveiled new functions of this interaction, i.e., promotion of phase separation of U2AF1 and modulation of mRNA splicing. Our work also established, for the first time, a biochemical basis for U2AF1's occupancy in gene promoters. Overall design: Genome-wide profiling analysis of POLR2A in wildtype HEK293T cells and U2AF1 knockdown or DHX9 knockdown HEK293T cells