Metabolic turnover and dynamics of modified ribonucleosides by 13C labeling

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 (m6A) 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 m62A-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.