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

Heiss, Matthias; Hagelskamp, Felix; Marchand, Virginie; Motorin, Yuri und Kellner, Stefanie (2021): Cell culture NAIL-MS allows insight into human tRNA and rRNA modification dynamics in vivo. In: Nature Communications, Bd. 12, Nr. 1, 389

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 tRNA(Phe) 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. Post transcriptional modification of RNAs represents an important layer of gene regulation. Here the authors describe NAIL-MS-a method for monoisotopic RNA labeling in cell culture-demonstrating its capabilities by analyzing the modification kinetics of total tRNA, 18S rRNA and tRNA(Phe) as models.

• Dokumententyp: Zeitschriftenartikel
• Fakultät: Chemie und Pharmazie
• Themengebiete: 500 Naturwissenschaften und Mathematik > 540 Chemie
• Sprache: Englisch