High-throughput quantitation of protein-RNA UV-crosslinking efficiencies as a predictive tool for high confidence identification of RNA binding proteins.

UV-crosslinking has proven to be an invaluable tool for the identification of RNA-protein interactomes. The paucity of methods for distinguishing background from bona fide RNA-protein interactions however makes attribution of RNA binding function on UV-crosslinking alone challenging. To address this need, we previously reported an RNA binding protein (RBP) confidence scoring metric, (RCS), incorporating both signal-to-noise (S:N) and protein abundance determinations to distinguish high and low confidence candidate RBPs. Although RCS has utility, we sought a direct metric for quantification and comparative evaluation of protein-RNA interactions. Here we propose the use of protein-specific UV-crosslinking efficiency (%CL), representing the molar fraction of a protein that is crosslinked to RNA, for functional evaluation of candidate RBPs. Application to the HeLa RNA interactome yielded %CL values for 1,097 proteins. Remarkably, %CL values span over five orders of magnitude. For the HeLa RNA interactome, %CL values comprise a range from high efficiency, high specificity interactions, e.g., the Elav protein HuR and the Pumilio homolog Pum2, with %CL values of 45.9 and 24.2, respectively, to very low efficiency and specificity interactions e.g., the metabolic enzymes glyceraldehyde-3-phosphate dehydrogenase, fructose-bisphosphate aldolase, and alpha-enolase, with %CL values of 0.0016, 0.006, and 0.008, respectively. We further extend the utility of %CL through prediction of protein domains and classes with known RNA-binding functions, thus establishing it as a useful metric for RNA interactome analysis. We anticipate that this approach will benefit efforts to establish functional RNA interactomes and support development of more predictive computational approaches for RNA binding protein identification.