# Identification of Amino Acids in Trm734 Required for 2′-O-Methylation of the tRNAPhe Wobble Residue

## TL;DR

This study identifies key amino acids in the Trm734 protein that are important for tRNA modification in yeast, which could help understand human diseases linked to these modifications.

## Contribution

The paper identifies specific amino acid regions in Trm734 critical for tRNA modification activity, not directly involved in protein-protein interactions.

## Key findings

- Key amino acids in Trm734 important for tRNA modification are located near the active site of Trm7.
- These residues are not involved in Trm7–Trm734 protein–protein interactions.
- A nonfunctional but stable Trm734 variant was discovered for further study of its cellular roles.

## Abstract

All organisms methylate
their nucleic acids, and this
methylation
is critical for proper gene expression at both the transcriptional
and translational levels. For proper translation in eukaryotes, 2′-O-methylation of C32 (Cm32) and G34 (Gm34) in the anticodon loop of tRNAPhe is critical, with defects in these modifications associated with
human disease. In yeast, Cm32 is formed by an enzyme that
consists of the methyltransferase Trm7 in complex with the auxiliary
protein Trm732, and Gm34 is formed by an enzyme that consists
of Trm7 in complex with Trm734. The role of Trm732 and Trm734 in tRNA
modification is not fully understood, although previous studies have
suggested that Trm734 is important for tRNA binding. In this report,
we generated Trm734 variants and tested their ability to work with
Trm7 to modify tRNAPhe. Using this approach, we identified
several regions of amino acids that are important for Trm734 activity
and/or stability. Based on the previously determined Trm7–Trm734
crystal structure, these crucial amino acids are near the active site
of Trm7 and are not directly involved in Trm7–Trm734 protein–protein
interactions. Immunoprecipitation experiments with these Trm734 variants
and Trm7 confirm that these residues are not involved in Trm7–Trm734
binding. Further experiments should help determine if these residues
are important for tRNA binding or have another role in the modification
of the tRNA. Furthermore, our discovery of a nonfunctional, stable
Trm734 variant will be useful in determining if the reported roles
of Trm734 in other biological processes such as retromer processing
and resistance to Ty1 transposition are due to tRNA modification defects
or to other bona fide cellular roles of Trm734.