Determining the role of Trm7, Trm732, and Trm734 in tRNA binding

Abstract

Modifications of the tRNA anticodon loop are important to translation. Proteins Trm7, Trm732, and Trm734 work together to modify the anticodon loop of tRNAPhe. Trm7 plays a catalytic role in methylation activity, and Trm732 and Trm734 are predicted to bind and position tRNAPhe for methylation at nucleotides C32 and G34 in Saccharomyces cerevisiae. Lack of Trm7 causes a sick phenotype in S.cerevisiae, and mutations in the TRM7 human ortholog, FTSJI, cause non-syndromic X-linked intellectual disability. We are working to determine whether individual Trm7, Trm732, and Trm734 proteins can bind to tRNAPhe, or whether Trm7:Trm732 and Trm7:Trm734 complexes are required for binding. Tagged proteins of interest are pulled down and the RNA bound to the proteins is analyzed by Northern Blot. This approach will also allow us to determine if our previously identified non-functional variants of Trm732 and Trm734 are still able to bind to tRNAPhe. Due to the high conservation between the yeast and human proteins, the results will likely be applicable to the orthologous proteins in humans