Identification of a Trm732 Motif Required for 2'- O-methylation of the tRNA Anticodon Loop by Trm7

Abstract

Posttranscriptional tRNA modifications are essential for proper gene expression, and defects in the enzymes that perform tRNA modifications are associated with numerous human disorders. Throughout eukaryotes, 2'-O-methylation of residues 32 and 34 of the anticodon loop of tRNA is important for proper translation, and in humans, a lack of these modifications results in non-syndromic X-linked intellectual disability. In yeast, the methyltransferase Trm7 forms a complex with Trm732 to 2'-O-methylate tRNA residue 32 and with Trm734 to 2'-O-methylate tRNA residue 34. Trm732 and Trm734 are required for the methylation activity of Trm7, but the role of these auxiliary proteins is not clear. Additionally, Trm732 and Trm734 homologs are implicated in biological processes not directly related to translation, suggesting that these proteins may have additional cellular functions. To identify critical amino acids in Trm732, we generated variants and tested their ability to function in yeast cells. We identified a conserved RRSAGLP motif in the conserved DUF2428 domain of Trm732 that is required for tRNA modification activity by both yeast Trm732 and its human homolog, THADA. The identification of Trm732 variants that lack tRNA modification activity will help to determine if other biological functions ascribed to Trm732 and THADA are directly due to tRNA modification or to secondary effects due to other functions of these proteins.

Figure 1

Schematic of 2′-O-methylation of the anticodon loop of tRNA^Phe in yeast. In yeast, the Trm7–Trm732 complex forms Cm₃₂ on tRNA^Phe, and the Trm7–Trm734 complex forms Gm₃₄.

Figure 2

Motif 2 of Trm732 is required for Cm₃₂ formation on tRNA^Phe. (A) Schematic representation of the Trm732 sequence. Inset box is an amino acid alignment of regions of high sequence similarity between Trm732 proteins from eight eukaryotes. Arrows point to the amino acids changed in Trm732 variants tested in this study. (B) Several conserved amino acids in Trm732 are required for suppression of the slow growth of trm732Δ trm734Δ mutants. Indicated strains containing URA3 and LEU2 plasmids were grown overnight in SD – Leu medium, diluted to an OD600 of ∼0.5, serially diluted 10-fold, and then spotted on medium containing 5-FOA to select against the URA3 plasmid. Cells were grown for 2 days at 30 °C. (C) Conserved amino acids in Trm732 are required for Cm₃₂ formation on tRNA^Phe in yeast. Quantification of nucleosides by UPLC from tRNA^Phe purified from indicated yeast strains, (*) levels below the threshold of detection.

Figure 3

Requirement of individual motif 2 residues for Trm732 function. (A) Amino acid residue R748 is important for Trm732 function. Strains with plasmids as indicated were grown overnight in SD-Leu and analyzed as in Figure 2B, after incubation for 2 days at 30 °C. In the top panel, following growth on 5-FOA at 30 °C, cells were spotted on YPD at 25 °C and incubated for 2 days.

Figure 4

Human THADA requires motif 2 for complementation of the yeast trm732Δ mutant. Indicated strains were grown overnight in the S medium containing raffinose and galactose; diluted as in Figure 2B; spotted to medium containing raffinose, galactose, and 5-FOA; and then incubated for 3 days at 30 °C.