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Metabolic turnover and dynamics of modified ribonucleosides by ¹³C labeling

Gameiro, PA; Encheva, V; Dos Santos, MS; MacRae, JI; Ule, J; (2021) Metabolic turnover and dynamics of modified ribonucleosides by ¹³C labeling. Journal of Biological Chemistry , Article 101294. 10.1016/j.jbc.2021.101294. (In press). Green open access

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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 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 non-stationary conditions. These results open new directions to probe the presence and biological regulation of modifications in particular RNAs.

Type: Article
Title: Metabolic turnover and dynamics of modified ribonucleosides by ¹³C labeling
Location: United States
Open access status: An open access version is available from UCL Discovery
DOI: 10.1016/j.jbc.2021.101294
Publisher version: https://doi.org/10.1016/j.jbc.2021.101294
Language: English
Additional information: This is an Open Access article published under a Creative Commons Attribution 4.0 International (CC BY 4.0) Licence (https://creativecommons.org/licenses/by/4.0/).
Keywords: 7-methylguanosine, Isotopic labeling, N6,N6-dimethyladenosine, N6-methyladenosine, RNA modifications, RNA turnover, mass spectrometry, metabolic stress, metabolism, methylation dynamics
UCL classification: UCL
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > UCL Queen Square Institute of Neurology
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > UCL Queen Square Institute of Neurology > Department of Neuromuscular Diseases
URI: https://discovery.ucl.ac.uk/id/eprint/10136700
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