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- W2952811048 abstract "The post-transcriptional modification of tRNA at the wobble position is a universal process occurring in all domains of life. In eukaryotes, the wobble uridine of particular tRNAs is transformed to the 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) modification which is critical for proper mRNA decoding and protein translation. However, current methods to detect mcm5s2U are technically challenging and/or require specialized instrumental expertise. Here, we show that γ-toxin endonuclease from the yeast Kluyveromyces lactis can be used as a probe for assaying mcm5s2U status in the tRNA of diverse eukaryotic organisms ranging from protozoans to mammalian cells. The assay couples the mcm5s2U-dependent cleavage of tRNA by γ-toxin with standard molecular biology techniques such as northern blot analysis or quantitative PCR to monitor mcm5s2U levels in multiple tRNA isoacceptors. The results gained from the γ-toxin assay reveals the evolutionary conservation of the mcm5s2U modification across eukaryotic species. Moreover, we have used the γ-toxin assay to verify uncharacterized eukaryotic Trm9 and Trm112 homologs that catalyze the formation of mcm5s2U. These findings demonstrate the use of γ-toxin as a detection method to monitor mcm5s2U status in diverse eukaryotic cell types for cellular, genetic, and biochemical studies." @default.
- W2952811048 created "2019-06-27" @default.
- W2952811048 creator A5049959557 @default.
- W2952811048 creator A5051016650 @default.
- W2952811048 creator A5073318315 @default.
- W2952811048 date "2018-02-13" @default.
- W2952811048 modified "2023-10-16" @default.
- W2952811048 title "Monitoring the 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) modification in eukaryotic tRNAs via the γ-toxin endonuclease" @default.
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