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Metabolic turnover and dynamics of modified ribonucleosides by 13C labeling

Research output: Contribution to journalArticlepeer-review

Paulo A. Gameiro, Vesela Encheva, Mariana Silva Dos Santos, James I. Macrae, Jernej Ule

Original languageEnglish
Article number101294
JournalJournal of Biological Chemistry
Volume297
Issue number5
DOIs
Published1 Nov 2021

Bibliographical note

Funding Information: Acknowledgments—This project has received funding from Wellcome Trust (103760/Z/14/Z) and the European Research Council (ERC) under the European Union’s Seventh Framework Program (FP7/2007-2013)/ERC grant agreement ID: 617837. The Francis Crick Institute receives its core funding from Cancer Research UK (grant no. FC001002), the UK Medical Research Council (grant no. FC001002), and the Wellcome Trust (grant no. FC001002). Funding Information: Funding and additional information—P. A. G. was supported by a Marie Sklodowska-Curie individual fellowship (grant agreement ID: 701730). Publisher Copyright: © 2021 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology.

King's Authors

Abstract

Tandem mass spectrometry (MS/MS) is an accurate tool to assess modified ribonucleosides and their dynamics in mammalian cells. However, MS/MS quantification of lowly abundant modifications in non-ribosomal RNAs is unreliable, and the dynamic features of various modifications are poorly understood. Here, we developed a 13C labeling approach, called 13C-dynamods, to quantify the turnover of base modifications in newly transcribed RNA. This turnover-based approach helped to resolve mRNA from ncRNA modifications in purified RNA or free ribonucleoside samples and showed the distinct kinetics of the N6-methyladenosine (m6A) versus 7-methylguanosine (m7G) modification in polyA+-purified RNA. We uncovered that N6,N6-dimethyladenosine (m62A) exhibits distinct turnover in small RNAs and free ribonucleosides when compared to known m62A-modified large rRNAs. Finally, combined measurements of turnover and abundance of these modifications informed on the transcriptional versus posttranscriptional sensitivity of modified ncRNAs and mRNAs, respectively, to stress conditions. Thus, 13C-dynamods enables studies of the origin of modified RNAs at steady-state and subsequent dynamics under nonstationary conditions. These results open new directions to probe the presence and biological regulation of modifications in particular RNAs.

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