tRNA m7G methyltransferase Trm8p/Trm82p: Evidence linking activity to a growth phenotype and implicating Trm82p in maintaining levels of active Trm8p

We show that Saccharomyces cerevisiae strains lacking Trm8p/Trm82p tRNA m 7 G methyltransferase are temperature-sensitive in synthetic media containing glycerol. Bacterial TRM8 orthologs complement the growth defect of trm8 -Δ, trm82 -Δ, and trm8 -Δ trm82 -Δ double mutants, suggesting that bacteria employ a single subunit for Trm8p/Trm82p function. The growth phenotype of trm8 mutants correlates with lack of tRNA m 7 G methyltransferase activity in vitro and in vivo, based on analysis of 10 mutant alleles of trm8 and bacterial orthologs, and suggests that m 7 G modification is the cellular function important for growth. Initial examination of the roles of the yeast subunits shows that Trm8p has most of the functions required to effect m 7 G modification, and that a major role of Trm82p is to maintain cellular levels of Trm8p. Trm8p efficiently cross-links to pre-tRNA Phe in vitro in the presence or absence of Trm82p, in addition to its known residual tRNA m 7 G modification activity and its SAM-binding domain. Surprisingly, the levels of Trm8p, but not its mRNA, are severely reduced in a trm82 -Δ strain. Although Trm8p can be produced in the absence of Trm82p by deliberate overproduction, the resulting protein is inactive, suggesting that a second role of Trm82p is to stabilize Trm8p in an active conformation.