biostudies-literatureHuang RNational Key Research and Development ProjectsNational Natural Science Foundation of ChinaE3879-E3887https://www.ebi.ac.uk/biostudies/studies/S-EPMC5924899biostudies-literatureUnknown115(17)Transcriptome-wide identification of RNA-binding proteins (RBPs) is a prerequisite for understanding the posttranscriptional gene regulation networks. However, proteomic profiling of RBPs has been mostly limited to polyadenylated mRNA-binding proteins, leaving RBPs on nonpoly(A) RNAs, including most noncoding RNAs (ncRNAs) and pre-mRNAs, largely undiscovered. Here we present a click chemistry-assisted RNA interactome capture (CARIC) strategy, which enables unbiased identification of RBPs, independent of the polyadenylation state of RNAs. CARIC combines metabolic labeling of RNAs with an alkynyl uridine analog and in vivo RNA-protein photocross-linking, followed by click reaction with azide-biotin, affinity enrichment, and proteomic analysis. Applying CARIC, we identified 597 RBPs in HeLa cProceedings of the National Academy of Sciences of the United States of AmericaTranscriptome-wide discovery of coding and noncoding RNA-binding proteins.PMC592489921521003917532062016YFA050150021425204Han MMeng LChen XHuang RfalseTranscriptome-wide discovery of coding and noncoding RNA-binding proteins.Transcriptome-wide identification of RNA-binding proteins (RBPs) is a prerequisite for understanding the posttranscriptional gene regulation networks. However, proteomic profiling of RBPs has been mostly limited to polyadenylated mRNA-binding proteins, leaving RBPs on nonpoly(A) RNAs, including most noncoding RNAs (ncRNAs) and pre-mRNAs, largely undiscovered. Here we present a click chemistry-assisted RNA interactome capture (CARIC) strategy, which enables unbiased identification of RBPs, independent of the polyadenylation state of RNAs. CARIC combines metabolic labeling of RNAs with an alkynyl uridine analog and in vivo RNA-protein photocross-linking, followed by click reaction with azide-biotin, affinity enrichment, and proteomic analysis. Applying CARIC, we identified 597 RBPs in HeLa c2018-01-01T00:00:00Z2018 Apr2026-04-29T09:07:16.376Z2019-03-27T00:03:21ZS-EPMC59248992963641910.1073/pnas.1718406115