Comprehensive Identification of RNA-Binding Domains in Human Cells

TLDR

Mammalian cells contain over a thousand RNA‑binding proteins, many of which bind RNA via unknown mechanisms, and RNA‑binding sites frequently overlap enzymatic cores, protein–protein interaction domains, and intrinsically disordered regions enriched for post‑translational modifications. The study develops RBDmap to map native RNA‑binding sites of RBPs across the proteome. RBDmap uses proteome‑wide crosslinking and mass spectrometry to identify RNA‑binding sites of native RBPs. RBDmap identified 1,174 RNA‑binding sites in 529 HeLa RBPs, revealing that many sites cluster near catalytic or protein‑protein interaction domains, reside in intrinsically disordered regions, are enriched for post‑translational modifications, and are evolutionarily conserved with disease‑associated mutations, underscoring the functional importance of RNA‑binding domains.

Abstract

Highlights•Experimental generation of an atlas of RNA-binding sites (RBS) in human cells•RBS overlap with enzymatic cores and protein-protein interaction sites•About half of the total RBS map to disordered protein regions•RBS are enriched for phosphorylation, acetylation, and methylation sitesSummaryMammalian 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.Graphical abstract