Mutations in MTO2 Related to tRNA Modification Impair Mitochondrial Gene Expression and Protein Synthesis in the Presence of a Paromomycin Resistance Mutation in Mitochondrial 15 S rRNA

Abstract

Nuclear gene(s) have been shown to modulate the phenotypic expression of mitochondrial DNA mutations. We report here the identification and characterization of the yeast nuclear gene MTO2 encoding an evolutionarily conserved protein involved in mitochondrial tRNA modification. Interestingly, mto2 null mutants expressed a respiratory-deficient phenotype when coexisting with the C1409G mutation of mitochondrial 15 S rRNA at the very conservative site for human deafness-associated 12 S rRNA A1491G and C1409T mutations. Furthermore, the overall rate of mitochondrial translation was markedly reduced in a yeast mto2 strain in the wild type mitochondrial background, whereas mitochondrial protein synthesis was almost abolished in a yeast mto2 strain carrying the C1409G allele. The other interesting feature of mto2 mutants is the defective expression of mitochondrial genes, especially CYTB and COX1, but only when coexisting with the C1409G allele. These data strongly indicate that a product of MTO2 functionally interacts with the decoding region of 15 S rRNA, particularly at the site of the C1409G or A1491G mutation. In addition, we showed that yeast and human Mto2p localize in mitochondria. The isolated human MTO2 cDNA can partially restore the respiratory-deficient phenotype of yeast mto2 cells carrying the C1409G mutation. These functional conservations imply that human MTO2 may act as a modifier gene, modulating the phenotypic expression of the deafness-associated A1491G or C1409T mutation in mitochondrial 12 S rRNA.

Human Mitochondrial Leucyl-tRNA Synthetase Corrects Mitochondrial Dysfunctions Due to the tRNALeu(UUR) A3243G Mutation, Associated with Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-Like Symptoms and Diabetes

“…A typical mitochondrial staining pattern was observed with both antibodies, and superimposition of two panels showed complete overlap of the two patterns, demonstrating that human LARS2 localizes exclusively to mitochondria. This suggested that this protein, like other nucleus-encoding mitochondrial proteins such as MTO1, GTPBP3, and TRMU (29,30,49), was synthesized on cytoplasmic ribosomes and was imported into and then functioned in the mitochondrion.…”

“…Resultant constructs were transfected into 143B cells using the SuperFect transfection reagent (Qiagen, Inc.) according to the manufacturer's protocol. Immunofluorescence analysis was performed as detailed elsewhere (29,30,49).…”

Modification of the wobble uridine in bacterial and mitochondrial tRNAs reading NNA/NNG triplets of 2-codon boxes

“…101,102 Proteins TRMU, MTO1 and MSS1 are required for splicing, likely because they are needed for the translation of maturases. 99,100,103,104 Strikingly, effects of MTO1 inactivation are more severe than those of MSS1 inactivation on splicing and accumulation of certain mttRNAs, 100 which might explain why mitochondrial translation is affected more in MTO1 than in MSS1 mutants. These observations suggest that MTO1 and MSS1, apart from their shared role in mttRNA modification, may play independent roles in other cellular functions.…”

Characterization of Human GTPBP3, a GTP-Binding Protein Involved in Mitochondrial tRNA Modification

“…Therefore, the function of the GidA, MnmE, and MnmA family proteins seems to be evolutionarily conserved. Human homologues have also been identified and been found to localize in mitochondria (23,25,46,49,50). It should be noted, however, that in humans taurine is incorporated into mitochondrial tRNA Lys and tRNA Leu(UUR) in place of cmnm (44).…”

“…In yeast, the protein products of the nuclear genes MSS1, MTO1, and MTU1 (homologues of mnmE, gidA, and mnmA, respectively) localize in mitochondria, and their mutants are defective in modification of mitochondrial tRNA (8,9,46,49). Thus, mitochondrial tRNA Lys molecules isolated from yeast MTO1 and MSS1 deletion strains contain s 2 U instead of the cmnm 5 s 2 U found in the wild-type tRNA Lys molecules, whereas disruption of the MTU1 gene eliminates the 2-thio modification of mitochondrial tRNAs (Fig.…”