Abstract: Yeast Saccharomyces cerevisiae MTO2, MTO1 and MSS1 genes encoded highly conserved tRNA modifying enzymes for the biosynthesis of 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)U34), 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 9 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, 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)U34 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.