RNP-MaP: In-cell analysis of protein interaction networks defines functional hubs in RNA

Abstract

RNAs interact with networks of proteins to form complexes (RNPs) that govern many biological processes, but these networks are currently impossible to examine in a comprehensive way. We developed a live-cell chemical probing strategy for mapping protein interaction networks in any RNA with single-nucleotide resolution. This RNP-MaP strategy (RNP network analysis by mutational profiling) simultaneously detects binding by and cooperative interactions involving multiple proteins with single RNA molecules. RNP-MaP revealed that two structurally related, but sequence-divergent noncoding RNAs, RNase P and RMRP, share nearly identical RNP networks and, further, that protein interaction network hubs identify function-critical sites in these RNAs. RNP-MaP identified numerous protein interaction networks within the XIST long noncoding RNA that are conserved between mouse and human RNAs and distinguished communities of proteins that network together on XIST. RNP-MaP data show that the Xist E region is densely networked by protein interactions and that PTBP1, MATR3, and TIA1 proteins each interface with the XIST E region via two distinct interaction modes; and we find that the XIST E region is sufficient to mediate RNA foci formation in cells. RNP-MaP will enable discovery and mechanistic analysis of protein interaction networks across any RNA in cells.