Insights into RNA Biology from an Atlas of Mammalian mRNA-Binding Proteins

Abstract

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.

Enhanced NOLC1 promotes cell senescence and represses hepatocellular carcinoma cell proliferation by disturbing the organization of nucleolus

“…A previous report indicated that CSIG is a nonclassical RNA binding protein (Castello et al ., 2012); thus, we wondered whether CSIG binds to NOLC1 mRNA and whether the destabilizing effect of CSIG on NOLC1 mRNA was dependent on its mRNA binding ability. Definitely, our immunoprecipitation (R‐IP) results showed that the NOLC1 mRNA immunoprecipitated with CSIG was enriched compared with IgG (Fig.…”

The greatest catch: big game fishing for mRNA-bound proteins

“…The two recent studies from the Hentze and Landthaler laboratories have taken the RNA bait concept a step further by capturing proteins by covalent cross-linking to polyadenylated RNA, mainly corresponding to mRNAs, and thereby generated a global mRNA interactome in HeLa [2] or HEK-293 [3] cell culture systems. In both studies, RNA-binding proteins are physically cross-linked to mRNAs using 254 nM UV-C light [2] or 365 nM UV in conjunction with PARs [2,3].…”

“…In both studies, RNA-binding proteins are physically cross-linked to mRNAs using 254 nM UV-C light [2] or 365 nM UV in conjunction with PARs [2,3]. Next, cellular mRNA and the bound interactome are efficiently captured with oligo-dT-coated beads and purified under stringent conditions to eliminate contamination from non-cross-linked proteins, including those deriving from non-cross-linked protein-protein interactions.…”

“…RNA-binding proteins are covalently cross-linked to RNAs using 254 nM UV-C light [2] or 365 nM UV light in conjunction with photoactivatable ribonucleosides (PARs) such as 4-thiouridine (4SU) [2,3]. The RNA is used as bait in a pull-down with oligo-dT-coated beads and purified under stringent conditions to eliminate contamination from non-cross-linked proteins.…”

“…Here we discuss two recent studies from the laboratories of Matthias Hentze [2] and Markus Landthaler [3], which have now globally captured and defined the RBPs bound to mRNAs within cultured human cells. Both identify an unexpected wealth of RBPs, many of which were not previously known to interact with RNA, and greatly expand our understanding of the mRNA interactome.…”