Abstract
SummaryMammalian 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.
Highlights
RNA metabolism relies on the dynamic interplay of RNAs with RNA-binding proteins (RBPs) forming ribonucleoprotein complexes, which control RNA fate from synthesis to decay (Glisovic et al, 2008)
Proteome-wide Mapping of RNA-binding domains (RBDs) by RBDmap To define how RNAbinding proteins (RBPs) bind to RNA in living cells, we extended RNA interactome capture (Castello et al, 2013b) by addition of an analytical protease digestion step followed by a second round of oligo(dT) capture and mass spectrometry (Figure 1A)
UV light is applied to cell monolayers to covalently stabilize native protein-RNA interactions taking place at ‘‘zero’’ distance (Pashev et al, 1991)
Summary
Highlights d Experimental generation of an atlas of RNA-binding sites (RBS) in human cells. Many recently discovered RNA-binding proteins (RBPs) do not show architectural similarities with classical RBPs, and their modes of interaction with RNA were unclear. We developed and employed RBDmap as a method for the comprehensive determination of the RNA-interacting sites of RBPs, identifying more than a thousand such sites. These data yield unprecedented insight into RNA-protein interactions in cells with implications for numerous biological contexts. 2016, Molecular Cell 63, 696–710 August 18, 2016 a 2016 The Author(s).
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