Post-transcriptional modifications of eukaryotic tRNA are crucial for proper cellular function. In Saccharomyces cerevisiae, the methyltransferase Trm7 is responsible for 2’-O-methylation of nucleotides 32 and 34 (Nm32 and Nm34), tRNA modifications associated with normal growth. Trm7 cannot function independently; it requires coupled activity with the proteins Trm732 and Trm734 to put on Nm32 and Nm34, respectively. FTSJ1, the ortholog of Trm7, is responsible for all tested Nm32 and Nm34 modifications in Homo sapiens. We aim to develop a reliable method for detection of 2’-O-methylations at varying positions on tRNA of metazoans. An ideal method would be highly sensitive and utilize minimal material. Primer extension of partially base hydrolyzed RNA has been used with 32P-labeled primers to detect Nm32 in S. cerevisiae, which suggests this approach could be used to further study other 2’-O-methylations in higher eukaryotes as well. Efforts are underway to use this method and others to detect 2’-O-methylations in cultured human and Drosophila melanogaster cells. To test for the requirement of TRM7, TRM732, and TRM734 for Nm32 and Nm34 in D. melanogaster, knockdown cells have been generated using RNAi from which bulk RNA can be extracted for partial base hydrolysis and primer extension.

Identification of tRNA 2'-O-methylation enzymes in humans and fruit flies

255th National Meeting (2018)

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