Post-transcriptional modifications are abundant in, and are required for, the proper function of tRNAs. 2’-O-methylation of tRNA residues 32 (Nm32) and 34 (Nm34) is conserved among eukaryotes, including yeast and humans. Trm7 methyltransferase interacts separately with Trm732 and Trm734 to form Cm32 and Gm34 on tRNAPhe, respectively. Trm7 and FTSJ1 have both been shown to interact with THADA to modify C32 in yeast. Mutations in FTSJ1 (human Trm7) cause intellectual disability and mutations in THADA (human Trm732) have been linked to polycystic ovary syndrome and type 2 diabetes. The exact function of Trm732 is currently unknown, but we are analyzing the conserved DUF2428 domain within Trm732 to help determine the role of the protein. Site-directed mutagenesis was used to generate variants within the DUF2428 domain to identify regions important for Trm732 function. Preliminary qualitative results indicate three important motifs important for Trm732 function: RRS748, GLP752, and RH701. Once the conditions were optimized for accurate Cm quantification, Ultra-Pressure Liquid Chromatography (UPLC) was utilized to analyze the Cm levels in each variant. In comparing our quantitative and qualitative results, we determined that the variant Cm levels correlate with decreased function of Trm732. We will be continuing to analyze the function of Trm732 in the methyltransferase reaction, both biochemically and genetically. tRNATrp and tRNALeu(UAA) will also be purified from each variant to quantify the Cm levels via UPLC. Binding between Trm7, Trm732, and tRNA will also be analyzed using yeast-2-hybrid and immunoprecipitation.

Identification of conserved motifs important for Trm732 function

2019 Spring National Meeting

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