TY - JOUR T1 - Cell culture NAIL-MS allows insight into human RNA modification dynamics <em>in vivo</em> JF - bioRxiv DO - 10.1101/2020.04.28.067314 SP - 2020.04.28.067314 AU - Matthias Heiss AU - Felix Hagelskamp AU - Stefanie Kellner Y1 - 2020/01/01 UR - http://biorxiv.org/content/early/2020/04/30/2020.04.28.067314.abstract N2 - In the last years, studies about the dynamics of RNA modifications are among the most controversially discussed. As the main reason, we have identified the unavailability of a technique which allows to follow the temporal dynamics of RNA transcripts in human cell culture. Here, we present a NAIL-MS (nucleic acid isotope labeling coupled mass spectrometry) scheme for efficient stable isotope labeling in both RNA and DNA (&gt;95% within 7 days) in common human cell lines and growth media. Validation experiments reveal that the labeling procedure itself does neither interfere with the isotope dilution MS quantification nor with RNA modification density. We design pulse chase NAIL-MS experiments and apply the new tool to study the temporal placement of modified nucleosides in e.g. tRNAPhe and 18S rRNA. In existing RNAs, we observe a constant loss of modified nucleosides over time which is masked by a post-transcriptional methylation mechanism and thus not detectable without NAIL-MS. During alkylation stress, NAIL-MS reveals an adaptation of tRNA modifications in new transcripts but not existing transcripts.Overall, we present a fast and reliable stable isotope labeling strategy which allows a more detailed study of RNA modification dynamics in human cell culture. With cell culture NAIL-MS it is finally possible to study the speed of both modification and demethylation reactions inside human cells. Thus it will be possible to study the impact of external stimuli and stress on human RNA modification kinetics and processing of mature RNA.Competing Interest StatementThe authors have declared no competing interest. ER -