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bioRxiv · 10.1101/2021.07.20.453046

Metabolic turnover and dynamics of modified ribonucleosides by 13C labeling

Gameiro, P. A.·Encheva, V.·Silva dos Santos, M.·MacRae, J. I.·Ule, J.
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Abstract

Tandem mass spectrometry (MS/MS) is an accurate tool to assess modified ribonucleosides and their dynamics in mammalian cells. Yet, MS/MS quantification of lowly abundant modifications in non-ribosomal RNAs is unreliable, and the dynamic features of various modifications poorly understood. We developed a 13C labeling approach, 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 ribonucleosides, and showed the distinct kinetics of N6-methyladenosine (m62A) versus 7-methylguanosine (m7G) in polyA+-purified RNA. We uncovered that N6,N6-dimethyladenosine (m62A) exhibits a distinct turnover in small RNAs and free ribonucleosides when compared to the known m6 A-modified large rRNAs. Finally, combined measurements of turnover and abundance informed on the transcriptional versus posttranscriptional sensitivity of modified ncRNAs and mRNAs, respectively, to stress conditions. Thus, 13C-dynamods enables studies of origin of modified RNAs at steady-state and their dynamics under non-stationary conditions.

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Gameiro, P. A., Encheva, V., Silva dos Santos, M., MacRae, J. I., Ule, J.. 2021-07-20. Metabolic turnover and dynamics of modified ribonucleosides by 13C labeling. https://doi.org/10.1101/2021.07.20.453046

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