Cell culture NAIL-MS allows insight into human tRNA and rRNA modification dynamics in vivo

Matthias Heiss, Felix Hagelskamp, Virginie Marchand, Yuri Motorin and Stefanie Kellner ()
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Matthias Heiss: Ludwig-Maximilians-University Munich
Felix Hagelskamp: Ludwig-Maximilians-University Munich
Virginie Marchand: Université de Lorraine, CNRS, Inserm, UMS2008/US40 IBSLor and UMR7365 IMoPA
Yuri Motorin: Université de Lorraine, CNRS, Inserm, UMS2008/US40 IBSLor and UMR7365 IMoPA
Stefanie Kellner: Ludwig-Maximilians-University Munich

Nature Communications, 2021, vol. 12, issue 1, 1-12

Abstract: Abstract Recently, studies about RNA modification dynamics in human RNAs are among the most controversially discussed. As a main reason, we identified the unavailability of a technique which allows the investigation of the temporal processing of RNA transcripts. Here, we present nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS) for efficient, monoisotopic stable isotope labeling in both RNA and DNA in standard cell culture. We design pulse chase experiments and study the temporal placement of modified nucleosides in tRNAPhe and 18S rRNA. In existing RNAs, we observe a time-dependent constant loss of modified nucleosides which is masked by post-transcriptional methylation mechanisms and thus undetectable without NAIL-MS. During alkylation stress, NAIL-MS reveals an adaptation of tRNA modifications in new transcripts but not existing ones. Overall, we present a fast and reliable stable isotope labeling strategy which allows in-depth study of RNA modification dynamics in human cell culture.

Date: 2021
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DOI: 10.1038/s41467-020-20576-4

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