1. NAIL‐MS in E. coli Determines the Source and Fate of Methylation in tRNA
- Author
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Verena Weber, Stefanie Kellner, and Valentin F. Reichle
- Subjects
0301 basic medicine ,Methylation ,Biochemistry ,Mass Spectrometry ,03 medical and health sciences ,chemistry.chemical_compound ,RNA, Transfer ,Very Important Paper ,In vivo ,Escherichia coli ,isotopic labeling ,tRNA ,Molecular Biology ,Demethylation ,TRNA methylation ,Full Paper ,Nitrogen Isotopes ,Chemistry ,Organic Chemistry ,RNA ,Full Papers ,Deuterium ,RNA demethylation ,Methyl methanesulfonate ,030104 developmental biology ,Isotope Labeling ,Transfer RNA ,Nucleic acid ,Molecular Medicine ,Ribonucleosides ,nucleosides - Abstract
In all domains of life, the nucleobases of tRNA can be methylated. These methylations are introduced either by enzymes or by the reaction of methylating agents with the nucleophilic centers of the nucleobases. Herein, we present a systematic approach to identify the methylation sites within RNA in vitro and in vivo. For discrimination between enzymatic tRNA methylation and tRNA methylation damage in bacteria, we used nucleic acid isotope labeling coupled mass spectrometry (NAIL‐MS). With NAIL‐MS, we clearly observed the formation of 7‐methylguanosine, 3‐methyluridine, and 6‐methyladenosine during exposure of bacteria to the alkylating agent methyl methanesulfonate (MMS) in vivo. These damage products were not reported to form in tRNA in vivo, as they were masked by the enzymatically formed modified nucleosides in previous studies. In addition, we found formation of the known damage products 1‐methyladenosine and 3‐methylcytidine in vivo. With a dynamic NAIL‐MS setup, we observed tRNA repair by demethylation of these two RNA modifications in vivo. Furthermore, we saw the potential repair of 6‐methyladenosine but not 7‐methylguanosine in bacterial tRNA.
- Published
- 2018