Combination of the loss of cmnm5U34 with the lack of s2U34 modifications of tRNALys, tRNAGlu, and tRNAGln altered mitochondrial biogenesis and respiration.

Yeast Saccharomyces cerevisiae MTO2, MTO1, and MSS1 genes encoded highly conserved tRNA modifying enzymes for the biosynthesis of carboxymethylaminomethyl (cmnm)(5)s(2)U(34) in mitochondrial tRNA(Lys), tRNA(Glu), and tRNA(Gln). In fact, Mto1p and Mss1p are involved in the biosynthesis of the cmnm(5) group (cmnm(5)U(34)), while Mto2p is responsible for the 2-thiouridylation (s(2)U(34)) of these tRNAs. Previous studies showed that partial modifications at U(34) in mitochondrial tRNA enabled mto1, mto2, and mss1 strains to respire. In this report, we investigated the functional interaction between MTO2, MTO1, and MSS1 genes by using the mto2, mto1, and mss1 single, double, and triple mutants. Strikingly, the deletion of MTO2 was synthetically lethal with a mutation of MSS1 or deletion of MTO1 on medium containing glycerol but not on medium containing glucose. Interestingly, there were no detectable levels of nine tRNAs including tRNA(Lys), tRNA(Glu), and tRNA(Gln) in mto2/mss1, mto2/mto1, and mto2/mto1/mss1 strains. Furthermore, mto2/mss1, mto2/mto1, and mto2/mto1/mss1 mutants exhibited extremely low levels of COX1 and CYTB mRNA and 15S and 21S rRNA as well as the complete loss of mitochondrial protein synthesis. The synthetic enhancement combinations likely resulted from the completely abolished modification at U(34) of tRNA(Lys), tRNA(Glu), and tRNA(Gln), caused by the combination of eliminating the 2-thiouridylation by the mto2 mutation with the absence of the cmnm(5)U(34) by the mto1 or mss1 mutation. The complete loss of modifications at U(34) of tRNAs altered mitochondrial RNA metabolisms, causing a degradation of mitochondrial tRNA, mRNA, and rRNAs. As a result, failures in mitochondrial RNA metabolisms were responsible for the complete loss of mitochondrial translation. Consequently, defects in mitochondrial protein synthesis caused the instability of their mitochondrial genomes, thus producing the respiratory-deficient phenotypes. Therefore, our findings demonstrated a critical role of modifications at U(34) of tRNA(Lys), tRNA(Glu), and tRNA(Gln) in maintenance of mitochondrial genome, mitochondrial RNA stability, translation, and respiratory function. Copyright 2009 Elsevier Ltd. All rights reserved.