Cell- and Polymerase-Selective Metabolic Labeling of Cellular RNA with 2'-Azidocytidine.

TitleCell- and Polymerase-Selective Metabolic Labeling of Cellular RNA with 2'-Azidocytidine.
Publication TypeJournal Article
Year of Publication2020
AuthorsWang, D, Zhang, Y, Kleiner, RE
JournalJ Am Chem Soc
Date Published2020 Aug 26
KeywordsCytidine, DNA-Directed RNA Polymerases, HeLa Cells, Humans, RNA

<p>Metabolic labeling of cellular RNA is a powerful approach to investigate RNA biology. In addition to revealing whole transcriptome dynamics, targeted labeling strategies can be used to study individual RNA subpopulations within complex systems. Here, we describe a strategy for cell- and polymerase-selective RNA labeling with 2'-azidocytidine (2'-AzCyd), a modified nucleoside amenable to bioorthogonal labeling with SPAAC chemistry. In contrast to 2'-OH-containing pyrimidine ribonucleosides, which rely upon uridine-cytidine kinase 2 (UCK2) for activation, 2'-AzCyd is phosphorylated by deoxycytidine kinase (dCK), and we find that expression of dCK mediates cell-selective 2'-AzCyd labeling. Further, 2'-AzCyd is primarily incorporated into rRNA and displays low cytotoxicity and high labeling efficiency. We apply our system to analyze the turnover of rRNA during ribophagy induced by oxidative stress or mTOR inhibition to show that 28S and 18S rRNAs undergo accelerated degradation. Taken together, our work provides a general approach for studying dynamic RNA behavior with cell and polymerase specificity and reveals fundamental insights into nucleotide and nucleic acid metabolism.</p>

Alternate JournalJ Am Chem Soc
PubMed ID32786764
PubMed Central IDPMC7720414
Grant ListR01 GM132189 / GM / NIGMS NIH HHS / United States