Strategies for Identifying Important Residues in the tRNA Modification Protein Trm732

Abstract

Post‐transcriptional tRNA modifications are required for efficient protein translation. Proteins involved in forming tRNA modifications are being studied to understand their role and function. In yeast, the Trm7 methyltransferase forms a complex with Trm732 to modify tRNA at position 32. In humans, lack of the Trm7 homolog, FTSJ1, has been linked to intellectual disabilities. The human Trm732 homolog, THADA, is associated with type 2 diabetes, PCOS, and cancers. Little is known about the function of Trm732, although we have shown that one conserved motif in Trm732 is important for tRNA modification. We are identifying other residues important for function using two strategies. First, we compare Trm732 proteins of different organisms using protein alignments to determine conserved regions to mutate for testing. Using this strategy, AIN801 was determined to be an important region for the function of Trm732 and we are continuing to look at other regions that may be of importance. Second, we are expressing randomly mutated Trm732 variants in a sick strain that lacks Trm732. Lack of rescue of strain indicates that the mutation present is harmful to protein function. DNA from these colonies is extracted and sequenced to determine the mutation that causes loss of Trm732 function. Understanding the functions of Trm7 and Trm732 in yeast could aid in our understanding of their human homologs, potentially providing insight into the causes of diseases.