capture-identification-rna-binding-proteins-using-click-chemistry

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture (CARIC) Strategy

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used ...

Peking University

The unexpected, non-enzymatic S-glycosylation of cysteine residues in various proteins by per-O-acetylated monosaccharides is described. This artificial S-glycosylation greatly compromises the specificity and validity of metabolic glycan labeling in living cells by per-O-acetylated azido and alkynyl sugars, which has been overlooked in the field for decades. It is demonstrated that the use of unacetylated unnatural sugars can avoid the artifact formation and a corrected list of O-GlcNAcylated proteins and O-GlcNAc sites in HeLa cells has been assembled by using N-azidoacetylgalactosamine (GalNAz).

Artificial Cysteine S-Glycosylation Induced by Per-O-Acetylated Unnatural Monosaccharides during Metabolic Glycan Labeling.

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 cells, including 130 previously unknown RBPs. These newly discovered RBPs can likely bind ncRNAs, thus uncovering potential involvement of ncRNAs in processes previously unknown to be ncRNA-related, such as proteasome function and intermediary metabolism. The CARIC strategy should be broadly applicable across various organisms to complete the census of RBPs.

Article	Year
Artificial Cysteine S-Glycosylation Induced by Per-O-Acetylated Unnatural Monosaccharides during Metabolic Glycan Labeling. Angewandte Chemie (International ed. in English)\| PubMed ID: 29237092	2018
Transcriptome-wide discovery of coding and noncoding RNA-binding proteins. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 29636419	2018

Mengting Han

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