Quantitative time-resolved chemoproteomics reveals that stable <i>O</i>-GlcNAc regulates box C/D snoRNP biogenesis.

Abstract

<i>O</i>-linked GlcNAcylation (<i>O</i>-GlcNAcylation), a ubiquitous posttranslational modification on intracellular proteins, is dynamically regulated in cells. To analyze the turnover dynamics of <i>O</i>-GlcNAcylated proteins, we developed a quantitative time-resolved <i>O</i>-linked GlcNAc proteomics (qTOP) strategy based on metabolic pulse-chase labeling with an <i>O</i>-GlcNAc chemical reporter and stable isotope labeling with amino acids in cell culture (SILAC). Applying qTOP, we quantified the turnover rates of 533 <i>O</i>-GlcNAcylated proteins in NIH 3T3 cells and discovered that about 14% exhibited minimal removal of <i>O</i>-GlcNAc or degradation of protein backbones. The stability of those hyperstable <i>O</i>-GlcNAcylated proteins was more sensitive to <i>O</i>-GlcNAcylation inhibition compared with the more dynamic populations. Among the hyperstable population were three core proteins of box C/D small nucleolar ribonucleoprotein complexes (snoRNPs): fibrillarin (FBL), nucleolar protein 5A (NOP56), and nucleolar protein 5 (NOP58). We showed that <i>O</i>-GlcNAcylation stabilized these proteins and was essential for snoRNP assembly. Blocking <i>O</i>-GlcNAcylation on FBL altered the 2'-<i>O</i>-methylation of rRNAs and impaired cancer cell proliferation and tumor formation in vivo.