RNA methylation regulates various aspects of RNA metabolism, and dynamic modulation of
RNA modifications has emerged as a major effector in cellular transitions. Yet, we lack quantitative
methods to comprehensively assess methylation dynamics, its features and regulatory inputs, across
RNA modifications. We developed 13C-dynamods, an isotopic labelling approach using [13C-methyl]-methionine, to quantify the turnover of base modifications in newly synthesized RNA. This turnover-based
approach resolved the contributions of mRNA vs. ncRNA modifications within polyadenylated
RNA and uncovered the distinct kinetics of N6-methyladenosine (m6A) and 7-methylguanosine (m7G)
in mRNA. Moreover, we obtained converging evidence indicating presence of N6,N6-
dimethyladenosine (m62A) in non-ribosomal RNA, in particular tRNA and rapidly decaying RNAs.
Finally, we showed that mRNA methylation dynamics is coordinated with ribonucleotide biosynthesis
during T-cell activation, and revealed post-transcriptional lability of m6A upon metabolic stress. Thus,
13C-dynamods enables studies of origin, maintenance and regulation of RNA modifications under
steady-state and non-stationary conditions.