Transfer RNAs (tRNAs) are molecules that transport amino acids to the ribosome to facilitate protein synthesis. Post-transcriptional modifications of tRNA are critical for proper functioning. Trm7 is a methyltransferase enzyme which interacts with Trm732 and Trm734 to modify tRNAs at residues C32 and G34, respectively. Trm7 and its accessory proteins are highly conserved amongst eukaryotes, including yeast and humans. Yeast mutants lacking both Trm732 and Trm734 are sick, indicating that these proteins are required for proper cell growth. Variations in the human homolog of Trm732, THADA, have been linked to polycystic ovary syndrome and type 2 diabetes. To better understand the function of Trm732, variants have been generated via site-directed mutagenesis and transformed into Saccharomyces cerevisiae. Analysis of these variants, when compared to wild type, allows for the identification of the Trm732 residues involved in the methylation of tRNA. Function has been investigated quantitatively by measuring tRNA nucleoside modification levels with Ultra-Pressure Liquid Chromatography, and qualitatively through growth assays. After studies of Trm732 in S. cerevisiae are complete, we will see if our findings translate to THADA in human cells. We have begun to analyze THADA knockouts in the HAP1 human cell line; tRNAPhe has been isolated from these cells and C32 modification levels are being measured. Quantification of Cm levels will provide information about the function of THADA in human cells.

Identification of residues required for Trm732 tRNA modification activity

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

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