Temporal resolution of NAIL-MS of tRNA, rRNA and Poly-A RNA is overcome by actinomycin D

RNA is dynamically modified and has the potential to respond to environmental changes and tune translation. The objective of this work is to uncover the temporal limitation of our recently developed cell culture NAIL-MS (nucleic acid isotope labelling coupled mass spectrometry) technology and overcome it. Actinomycin D (AcmD), an inhibitor of transcription, was used in the NAIL-MS context to reveal the origin of hybrid nucleoside signals composed of unlabelled nucleosides and labelled methylation marks. We find that the formation of these hybrid species depends exclusively on transcription for Poly-A RNA and rRNA but is partly transcription-independent for tRNA. This finding suggests that tRNA modifications adapt and are dynamically regulated by cells to overcome e.g. stress. Future studies on the tRNA modification mediated stress response are now accessible and the temporal resolution of NAIL-MS is improved by the use of AcmD. Metabolic labelling of RNA in human cell culture results in hybrid RNA species which can act to improve the temporal resolution of RNA modification dynamics studies.