Mammalian cells harbor more than a thousand RNA-binding proteins (RBPs), with half of these employing unknown modes of RNA binding. We developed RBDmap to determine the RNA-binding sites of native RBPs on a proteome-wide scale. We identified 1,174 binding sites within 529 HeLa cell RBPs, discovering numerous RNA-binding domains (RBDs). Catalytic centers or protein-protein interaction domains are in close relationship with RNA-binding sites, invoking possible effector roles of RNA in the control of protein function. Nearly half of the RNA-binding sites map to intrinsically disordered regions, uncovering unstructured domains as prevalent partners in protein-RNA interactions. RNA-binding sites represent hot spots for defined posttranslational modifications such as lysine acetylation and tyrosine phosphorylation, suggesting metabolic and signal-dependent regulation of RBP function. RBDs display a high degree of evolutionary conservation and incidence of Mendelian mutations, suggestive of important functional roles. RBDmap thus yields profound insights into native protein-RNA interactions in living cells
