Project description:RNAs interact with networks of proteins to form complexes (RNPs) that govern many biological processes, but inter-protein networks on RNA are currently impossible to examine in a comprehensive way. We developed a live-cell RNP-MaP (RNP network analysis by mutational profiling) chemical probing strategy for mapping simultaneous binding by and cooperative interactions among multiple proteins with single RNA molecules at nucleotide resolution. 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-mediated structural communication identifies function-critical network hubs in these RNAs. RNP-MaP identified previously unknown protein interaction networks within the XIST long noncoding RNA that are conserved between mouse and human RNAs and defined silencing, compartmentalization and splicing communities of proteins whose binding sites are networked together on XIST. The XIST E region contains a dense network of protein interactions, and including PTBP1, MATR3, and TIA1 proteins , which RNP-MaP revealed to each bind the XIST E region via two distinct interaction modes.; Depletion of PTBP1 and MATR3 caused native XIST particles to disperse and disappear in a human cell line. the The highly networked XIST E region was sufficient to mediate XIST RNA-like foci formation in cells. RNP-MaP enables discovery and prioritization of in-cell protein interaction networks critical for function in long RNAs, in the absence of pre-existing knowledge about protein binding sites.
Project description:Many proteins regulate the expression of genes by binding to specific regions encoded in the genome1. Here we introduce a new data set of RNA elements in the human genome that are recognized by RNA-binding proteins (RBPs), generated as part of the Encyclopedia of DNA Elements (ENCODE) project phase III. This class of regulatory elements functions only when transcribed into RNA, as they serve as the binding sites for RBPs that control post-transcriptional processes such as splicing, cleavage and polyadenylation, and the editing, localization, stability and translation of mRNAs. We describe the mapping and characterization of RNA elements recognized by a large collection of human RBPs in K562 and HepG2 cells. Integrative analyses using five assays identify RBP binding sites on RNA and chromatin in vivo, the in vitro binding preferences of RBPs, the function of RBP binding sites and the subcellular localization of RBPs, producing 1,223 replicated data sets for 356 RBPs. We describe the spectrum of RBP binding throughout the transcriptome and the connections between these interactions and various aspects of RNA biology, including RNA stability, splicing regulation and RNA localization. These data expand the catalogue of functional elements encoded in the human genome by the addition of a large set of elements that function at the RNA level by interacting with RBPs.