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Cell culture NAIL-MS allows insight into human tRNA and rRNA modification dynamics in vivo
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Cell culture NAIL-MS allows insight into human tRNA and rRNA modification dynamics in vivo
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Journal Article
Cell culture NAIL-MS allows insight into human tRNA and rRNA modification dynamics in vivo
2021
Overview
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.
Publisher
Nature Publishing Group UK,Nature Publishing Group,Nature Portfolio
Subject
/ 45/71
/ 45/90
/ 45/91
/ Biochemistry, Molecular Biology
/ Cell Culture Techniques - methods
/ DNA
/ Genomics
/ Humanities and Social Sciences
/ Humans
/ Kinetics
/ Labeling
/ RNA
/ RNA Processing, Post-Transcriptional - physiology
/ RNA, Ribosomal, 18S - chemistry
/ RNA, Ribosomal, 18S - metabolism
/ RNA, Transfer, Phe - chemistry
/ RNA, Transfer, Phe - metabolism
/ rRNA 18S
/ Science
/ tRNA
/ tRNA Ala
