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.

Exercise has a range of effects on metabolism. In animal models, repeated exertion reduces malignant tumour progression, and clinically, exercise can improve outcome for cancer patients. The etiology of the effect of exercise on tumour progression is unclear, as are the cellular actors involved. We show here that exercise-induced reduction in tumour growth is dependent on CD8+ T cells and that lactate, which is produced at high levels during exertion, increases proliferative capacity and cytotoxicity of CD8+ T cells. We found that at elevated levels lactate is used as a fuel during T cell activation. We further found that injection of lactate into animals can reduce malignant tumour growth in a dose-and CD8+ T cell-dependent manner. These data demonstrate that lactate can act to increase the anti-tumour activity of cytotoxic T cells, and in so doing, reduce cancer progression.

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 ¹³ C labeling approach, ¹³ C-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 N 6-methyladenosine (m ⁶ ₂ A) versus 7-methylguanosine (m ⁷ G) in polyA+-purified RNA. We uncovered that N 6, N 6-dimethyladenosine (m ⁶ ₂ A) exhibits a distinct turnover in small RNAs and free ribonucleosides when compared to the known m ⁶ 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, ¹³ C-dynamods enables studies of origin of modified RNAs at steady-state and their dynamics under non-stationary conditions.

RNA methylation regulates various aspects of RNA metabolism, and dynamic modulation ofRNA modifications has emerged as a major effector in cellular transitions. Yet, we lack quantitativemethods to comprehensively assess methylation dynamics, its features and regulatory inputs, acrossRNA 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-basedapproach resolved the contributions of mRNA vs. ncRNA modifications within polyadenylatedRNA 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 biosynthesisduring 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 understeady-state and non-stationary conditions.