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Study of Trm7 Interactions With Binding Partners Trm732 and Trm734 for 2’‐O‐Methylation of the tRNA Anticodon Loop in Yeast

Transfer RNAs undergo post‐transcriptional modifications, including many which occur at or near the anticodon loop and are necessary for protein synthesis. In Saccharomyces cerevisiae, Trm7 is required for 2’‐O‐methylation of tRNA and requires interaction with Trm732 and Trm734 for modifications at positions 32 and 34, respectively. These proteins are widely conserved in eukaryotes, and mutations in the human ortholog of TRM7, FTSJ1, can cause non‐syndromic X‐linked intellectual disability by decreasing or eliminating methylation activity. Through a genetic assay, we have found Trm7 residues required for interaction with Trm732 or Trm734 and the subsequent methylation of tRNAPhe. These spot test assays showed that the Trm7 Y138R variant abrogates interaction with Trm734 and the Trm7 I144A variant abrogates interaction with Trm732. To verify that these genetic results are due to impaired interaction of Trm7 variants with its binding partners, we are performing immunoprecipitation assays. In addition, we have begun to study interactions of the individual proteins and complexes with tRNAPhe through biochemical methods such as electrophoretic mobility shift assays, to determine the role of each protein in tRNA binding. Through this study of Trm7 variants, we can better understand the interaction of Trm7 with Trm734 and Trm732, which will facilitate the study of conserved homologs in humans.