Comprehensive Identification of RNA-Binding Domains in Human Cells

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.

The yeast noncoding RNA interaction network

“…As shown in Figure 7, some canonical RBDs, such as RRM, DEXDC, and S1, were enriched in comparison with the yeast proteome; however, most of the proteins do not contain any of them, but contain specific regions selectively found in single proteins (such as Nop10 domain in Nop10p, UniProtKB: Q6Q547; or Utp8 domain in Utp8p, UniProtKB: P53276; or Mpp10 domain in Mpp10p, UniProtKB: P47083; and so forth), or are composed of disordered regions only (such as SF3B1, UniProtKB: P49955 or UTP9, UniProtKB: P38882, or Nsa1, UniProtKB: P53136), suggesting the evolution of multiple RNA binding strategies. It has been recently reported that yeast mRNA binding proteins often lack typical RBDs (Beckmann et al 2015;MatiaGonzalez et al 2015;Brannan et al 2016;Castello et al 2016a). We compared the occurrence of the most common RNA binding domains in yeast ncRNA binding proteins (BP) versus a list of yeast mRNA BP (Mitchell et al 2013), and we observed that ncRNAs are recognized by unconventional modules similarly to or even more often than mRNAs (Fig.…”

High-Resolution Mapping of RNA-Binding Regions in the Nuclear Proteome of Embryonic Stem Cells

“…While our manuscript was being revised, Hentze and colleagues used a different technique to map RBRs in HeLa cells (Castello et al, 2016). Their approach relies on two sequential oligo-dT pull-downs and therefore might have lower false positive rates than RBR-ID; however, it can only be used to identify RBPs that bind polyA + RNA and requires 10–100 times more input material than RBR-ID.…”

Spatial patterning of P granules by RNA-induced phase separation of the intrinsically-disordered protein MEG-3

“…The region immediately C-terminal scores close to the IUPRED cut-off (Wang et al, 2014), and may contribute to RNA binding. IDRs are over-represented among RNA-binding domains (Varadi et al, 2015; Castello et al, 2016). Electrostatic interactions between positively-charged amino acids and the negatively-charged RNA backbone are often invoked as a possible mechanism for RNA binding by IDRs (Guo and Shorter, 2015; Basu and Bahadur, 2016).…”