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

RNA. 2024 Feb 29:rna.079848.123. doi: 10.1261/rna.079848.123. Online ahead of print.ABSTRACTUV-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...

Source: RNA - February 29, 2024 Category: Genetics & Stem Cells Authors: Johncarlo Kristofich Christopher Nicchitta Source Type: research