ABSTRACTThe 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 gamma-toxin endonuclease from the yeastKluyveromyces lactiscan 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 gamma-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 gamma-toxin assay reveals the evolutionary conservation of the mcm5s2U modification across eukaryotic species. Moreover, we have employed the gamma-toxin assay to verify uncharacterized eukaryotic Trm9 and Trm112 homologs that catalyze the formation of mcm5s2U. These findings demonstrate the use of gamma-toxin as a detection method to monitor mcm5s2U status in diverse eukaryotic cell types for cellular, genetic and biochemical studies.