Abstract 1339 Determination of the effect on translation of 2'-O-methylation at positions 32 and 34 in eukaryotic tRNA

Post-transcriptional modification of tRNA is vital for efficient protein translation, with many human diseases associated with modification defects.Mutations in the widely conserved eukaryotic methyltransferase TRM7 causes non-syndromic Xlinked intellectual disability in humans and slow growth in yeast.Yeast Trm7 interacts with Trm732 and Trm734 to perform 2'-O-methylation at C32 and G34, respectively, on tRNAPhe, and corresponding proteins are responsible for these modifications on human tRNA.In budding yeast, lack of 2'-O-methylation at both C32 and G34 causes growth defects, but if only one modification is missing, cells are still healthy.Consequently, the individual role of Cm32 and Gm34 is not clear.Furthermore, because both UUU and UUC code for Phe, it is not clear how each modification affects translation of each codon.Bidirectional expression of red and green fluorescent proteins (RFP and GFP) via the RNA-ID reporter system in yeast allows for the quantitative analysis of the effect of tRNA modifications on the translation of codons in cells.We have placed additional UUU and UUC codons at the start of the GFP, allowing us to test the effect of Cm32 and Gm34 on translation of these codons by determining the GFP/RFP ratio of reporters in trm734D and trm732D mutants, and then comparing those values to the reporters in wildtype strains.We are also determining the effect of UGG and UUA codons on GFP expression in these mutants, because these codons are decoded by other tRNAs that receive Nm32 and Nm34 modifications.Understanding how Trm7-dependent tRNA modifications affect translation in yeast will provide insights into how these modifications may affect translation in other eukaryotes, including humans.