RNA-binding proteins (RBPs) determine RNA fate from synthesis to decay. Employing two complementary protocols for covalent UV crosslinking of RBPs to RNA, we describe a systematic, unbiased, and comprehensive approach, termed "interactome capture," to define the mRNA interactome of proliferating human HeLa cells. We identify 860 proteins that qualify as RBPs by biochemical and statistical criteria, adding more than 300 RBPs to those previously known and shedding light on RBPs in disease, RNA-binding enzymes of intermediary metabolism, RNA-binding kinases, and RNA-binding architectures. Unexpectedly, we find that many proteins of the HeLa mRNA interactome are highly intrinsically disordered and enriched in short repetitive amino acid motifs. Interactome capture is broadly applicable to study mRNA interactome composition and dynamics in varied biological settings.

RNA-binding proteins (RBPs) exert a broad range of biological functions. To explore the scope of RBPs across eukaryotic evolution, we determined the in vivo RBP repertoire of the yeast Saccharomyces cerevisiae and identified 678 RBPs from yeast and additionally 729 RBPs from human hepatocytic HuH-7 cells. Combined analyses of these and recently published data sets define the core RBP repertoire conserved from yeast to man. Conserved RBPs harbour defined repetitive motifs within disordered regions, which display striking evolutionary expansion. Only 60% of yeast and 73% of the human RBPs have functions assigned to RNA biology or structural motifs known to convey RNA binding, and many intensively studied proteins surprisingly emerge as RBPs (termed ‘enigmRBPs'), including almost all glycolytic enzymes, pointing to emerging connections between gene regulation and metabolism. Analyses of the mitochondrial hydroxysteroid dehydrogenase (HSD17B10) uncover the RNA-binding specificity of an enigmRBP.

rnA-protein complexes play pivotal roles in many central biological processes. Although methods based on highthroughput sequencing have advanced our ability to identify the specific rnAs bound by a particular protein, there is a need for precise and systematic ways to identify rnA interaction sites on proteins. We have developed an experimental and computational workflow combining photo-induced crosslinking, high-resolution mass spectrometry and automated analysis of the resulting mass spectra for the identification of cross-linked peptides, cross-linking sites and the cross-linked rnA oligonucleotide moieties of such rnA-binding proteins. the workflow can be applied to any rnA-protein complex of interest or to whole proteomes. We applied the approach to human and yeast mrnA-protein complexes in vitro and in vivo, demonstrating its powerful utility by identifying 257 cross-linking sites on 124 distinct rnA-binding proteins. the open-source software pipeline developed for this purpose, rnP xl , is available as part of the openms project.RNA molecules bind to proteins to form ribonucleoprotein complexes (RNPs). These are indispensable for the synthesis, stability, transport and activity of mRNAs 1 and noncoding RNAs 2,3 . RNA-binding proteins (RBPs) assume numerous functions in RNPs. RBPs can modulate or stabilize RNA structures, thereby making RNA catalytically active, for example, during pre-mRNA splicing 4 . RNA can also guide a catalytically active RBP to its destination; examples of this are microRNA-or long noncoding RNA-mediated translational control and epigenetic modulation 5,6 . RBPs are also involved in splicing and can recruit or repel other proteins, induce hydrolysis of RNA or protect RNA from degradation.