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

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.     

Mutation in MTO1 involved in tRNA modification impairs mitochondrial RNA metabolism in the yeast Saccharomyces cerevisiae

The yeast MTO1 gene encodes an evolutionarily conserved protein for the biosynthesis of the 5-carboxymethylaminomethyl group of cmnm⁵s²U in the wobble position of mitochondrial tRNA. However, mto1 null mutant expressed the respiratory deficient phenotype only when coupled with the C1409G mutation of mitochondrial 15S rRNA. To further understand the role of MTO1 in mitochondrial RNA metabolism, the yeast mto1 null mutants carrying either wild-type (P^S) or 15S rRNA C1409G allele (P^R) have been characterized by examining the steady-state levels, aminoacylation capacity of mitochondrial tRNA, mitochondrial gene expression and petite formation. The steady-state levels of tRNA^Lys, tRNA^Glu, tRNA^Gln, tRNA^Leu, tRNA^Gly, tRNA^Arg and tRNA^Phe were decreased significantly while those of tRNA^Met and tRNA^His were not affected in the mto1 strains carrying the P^S allele. Strikingly, the combination of the mto1 and C1409G mutations gave rise to the synthetic phenotype for some of the tRNAs, especially in tRNA^Lys, tRNA^Met and tRNA^Phe. Furthermore, the mto1 strains exhibited a marked reduction in the aminoacylation levels of mitochondrial tRNA^Lys, tRNA^Leu, tRNA^Arg but almost no effect in those of tRNA^His. In addition, the steady-state levels of mitochondrial COX1, COX2, COX3, ATP6 and ATP9 mRNA were markedly decreased in mto1 strains. These data strongly indicate that unmodified tRNA caused by the deletion of MTO1 gene caused the instability of mitochondrial tRNAs and mRNAs and an impairment of aminoacylation of mitochondrial tRNAs. Consequently, the deletion of MTO1 gene acts in synergy with the 15S rRNA C1409G mutation, leading to the loss of COX1 synthesis and subsequent respiratory deficient phenotype.     

Isolation and characterization of the putative nuclear modifier gene MTO1 involved in the pathogenesis of deafness-associated mitochondrial 12 S rRNA A1555G mutation

The human mitochondrial 12 S rRNA A1555G mutation has been found to be associated with aminoglycoside-induced and non-syndromic deafness. However, putative nuclear modifier gene(s) have been proposed to regulate the phenotypic expression of this mutation. In yeast, the mutant alleles of MTO1, encoding a mitochondrial protein, manifest respiratory-deficient phenotype only when coupled with the mitochondrial 15 S rRNA P(R)454 mutation corresponding to human A1555G mutation. This suggests that the MTO1-like modifier gene may influence the phenotypic expression of human A1555G mutation. Here we report the identification of full-length cDNA and elucidation of genomic organization of the human MTO1 homolog. Human Mto1 is an evolutionarily conserved protein that implicates a role in the mitochondrial tRNA modification. Functional conservation of this protein is supported by the observation that isolated human MTO1 cDNA can complement the respiratory deficient phenotype of yeast mto1 cells carrying P(R)454 mutation. MTO1 is ubiquitously expressed in various tissues, but with a markedly elevated expression in tissues of high metabolic rates including cochlea. These observations suggest that human MTO1 is a structural and functional homolog of yeast MTO1. Thus, it may play an important role in the pathogenesis of deafness-associated A1555G mutation in 12 S rRNA gene or mutations in tRNA genes.     

Combination of the Loss of cmnmU34 with the Lack of sU34 Modifications of tRNA, tRNA, and tRNA 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)⁵s²U₃₄ in mitochondrial tRNA^Lys, tRNA^Glu, and tRNA^Gln. In fact, Mto1p and Mss1p are involved in the biosynthesis of the cmnm⁵ group (cmnm⁵U₃₄), while Mto2p is responsible for the 2-thiouridylation (s²U₃₄) of these tRNAs. Previous studies showed that partial modifications at U₃₄ 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 U₃₄ 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 cmnm⁵U₃₄ by the mto1 or mss1 mutation. The complete loss of modifications at U₃₄ 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 U₃₄ of tRNA^Lys, tRNA^Glu, and tRNA^Gln in maintenance of mitochondrial genome, mitochondrial RNA stability, translation, and respiratory function.     

Deletion of the MTO2 gene related to tRNA modification causes a failure in mitochondrial RNA metabolism in the yeast Saccharomyces cerevisiae

We report here the characterization of the yeast mto2 null mutants carrying wild-type mitochondrial DNA or 15S rRNA C1049G allele. The amounts of mitochondrial tRNA(Lys), tRNA(Glu), tRNA(Gln), tRNA(Leu), tRNA(Gly) and tRNA(Met) were markedly decreased but those of tRNA(Arg) and tRNA(His) were not affected in mto2 strains. The mto2 strains exhibited significant reduction in the aminoacylation of tRNA(Lys), tRNA(Leu) but almost no effect in those of tRNA(His). Interestingly, the strain carrying the C1049G allele exhibited an impairment of aminoacylation of those tRNAs. Furthermore, the steady-state levels of mitochondrial mRNA CYTB, COX1, COX2, COX3, and ATP6 were markedly decreased in mto2 strains. These data strongly indicate that unmodified tRNA caused by the deletion of MTO2 caused the instability of mitochondrial tRNAs and mRNAs and impairment of aminoacylation of tRNAs.     

Mutations of the mitochondrial-tRNA modifier MTO1 cause hypertrophic cardiomyopathy and lactic acidosis

Dysfunction of mitochondrial respiration is an increasingly recognized cause of isolated hypertrophic cardiomyopathy. To gain insight into the genetic origin of this condition, we used next-generation exome sequencing to identify mutations in MTO1, which encodes mitochondrial translation optimization 1. Two affected siblings carried a maternal c.1858dup (p.Arg620Lysfs^∗8) frameshift and a paternal c.1282G>A (p.Ala428Thr) missense mutation. A third unrelated individual was homozygous for the latter change. In both humans and yeast, MTO1 increases the accuracy and efficiency of mtDNA translation by catalyzing the 5-carboxymethylaminomethylation of the wobble uridine base in three mitochondrial tRNAs (mt-tRNAs). Accordingly, mutant muscle and fibroblasts showed variably combined reduction in mtDNA-dependent respiratory chain activities. Reduced respiration in mutant cells was corrected by expressing a wild-type MTO1 cDNA. Conversely, defective respiration of a yeast mto1Δ strain failed to be corrected by an Mto1^Pro622∗ variant, equivalent to human MTO1^{Arg620Lysfs∗8}, whereas incomplete correction was achieved by an Mto1^Ala431Thr variant, corresponding to human MTO1^Ala428Thr. The respiratory yeast phenotype was dramatically worsened in stress conditions and in the presence of a paromomycin-resistant (P^R) mitochondrial rRNA mutation. Lastly, in vivo mtDNA translation was impaired in the mutant yeast strains.     

Co-segregation of the T1095C with the A1555G mutation of the mitochondrial 12S rRNA gene in a patient with non-syndromic hearing loss

We reported here the clinical and molecular characterization of a Chinese subject with childhood-onset hearing impairment. Clinical evaluations showed that the patient suffered from profound and non-syndromic sensorineural hearing loss with flat configurations. Sequence analysis of the mitochondrial 12S rRNA and tRNA^Ser(UCN) genes led to the identification of double deafness-associated mutations of A1555G and T1095C in the 12S rRNA gene which apparently in the homoplasmic forms. In additional, there was no other functionally significant nucleotide variants found in this subject. As previous studies have indicated that the A1555G mutation was a primary contributing factor underlying the development of deafness but not sufficient to produce clinical phenotype, the co-segregation of two mitochondrial DNA mutations raises the possibility that the T to C transition at position 1095 plays a role in the phenotypic expression of deafness-associated A1555G mutation. Actually, the T1095C mutation disrupted an evolutionarily conserved base-pair at stem-loop of helix 25 of 12S rRNA, resulting in impaired translation in mitochondrial protein synthesis and a significant reduction of cytochrome c oxidase activity. As a result, it may enhance the biochemical defect in patient carrying the A1555G mutation, thus changing the age of onset and the severity of hearing impairment.     

Mitochondria-specific RNA-modifying enzymes responsible for the biosynthesis of the wobble base in mitochondrial tRNAs. Implications for the molecular pathogenesis of human mitochondrial diseases

Human mitochondrial (mt) tRNA(Lys) has a taurine-containing modified uridine, 5-taurinomethyl-2-thiouridine (taum5s2U), at its anticodon wobble position. We previously found that the mt tRNA(Lys), carrying the A8344G mutation from cells of patients with myoclonus epilepsy associated with ragged-red fibers (MERRF), lacks the taum5s2U modification. Here we describe the identification and characterization of a tRNA-modifying enzyme MTU1 (mitochondrial tRNA-specific 2-thiouridylase 1) that is responsible for the 2-thiolation of the wobble position in human and yeast mt tRNAs. Disruption of the yeast MTU1 gene eliminated the 2-thio modification of mt tRNAs and impaired mitochondrial protein synthesis, which led to reduced respiratory activity. Furthermore, when MTO1 or MSS1, which are responsible for the C5 substituent of the modified uridine, was disrupted along with MTU1, a much more severe reduction in mitochondrial activity was observed. Thus, the C5 and 2-thio modifications act synergistically in promoting efficient cognate codon decoding. Partial inactivation of MTU1 in HeLa cells by small interference RNA also reduced their oxygen consumption and resulted in mitochondria with defective membrane potentials, which are similar phenotypic features observed in MERRF.     

Nuclear factors: Roles related to mitochondrial deafness

Hearing loss (HL) is a common disorder with mitochondrial dysfunction as one of the major causes leading to deafness. Mitochondrial dysfunction may be caused by either mutations in nuclear genes leading to defective nuclear-encoded proteins or mutations in mitochondrial genes leading to defective mitochondrial-encoded products. The specific nuclear genes involved in HL can be classified into two categories depending on whether mitochondrial gene mutations co-exist (modifier genes) or not (deafness-causing genes). TFB1M, MTO1, GTPBP3, and TRMU are modifier genes. A mutation in any of these modifier genes may lead to a deafness phenotype when accompanied by the mitochondrial gene mutation. OPA1, TIMM8A, SMAC/DIABLO, MPV17, PDSS1, BCS1L, SUCLA2, C10ORF2, COX10, PLOG1and RRM2B are deafness-causing genes. A mutation in any of these deafness-causing genes will directly induce variable phenotypic HL.     

Maternally inherited aminoglycoside-induced and nonsyndromic deafness is associated with the novel C1494T mutation in the mitochondrial 12S rRNA gene in a large Chinese family

We report here the characterization of a large Chinese family with maternally transmitted aminoglycoside-induced and nonsyndromic deafness. In the absence of aminoglycosides, some matrilineal relatives in this family exhibited late-onset/progressive deafness, with a wide range of severity and age at onset. Notably, the average age at onset of deafness has changed from 55 years (generation II) to 10 years (generation IV). Clinical data reveal that the administration of aminoglycosides can induce or worsen deafness in matrilineal relatives. The age at the time of drug administration appears to be correlated with the severity of hearing loss experienced by affected individuals. Sequence analysis of mitochondrial DNA in this pedigree identified a homoplasmic C-to-T transition at position 1494 (C1494T) in the 12S rRNA gene. The C1494T mutation is expected to form a novel U1494-1555A base pair, which is in the same position as the C1494-1555G pair created by the deafness-associated A1555G mutation, at the highly conserved A site of 12S rRNA. Exposure to a high concentration of paromomycin or neomycin caused a variable but significant average increase in doubling time in lymphoblastoid cell lines derived from four symptomatic and two asymptomatic individuals in this family carrying the C1494T mutation when compared to four control cell lines. Furthermore, a significant decrease in the rate of total oxygen consumption was observed in the mutant cell lines. Thus, our data strongly support the idea that the A site of mitochondrial 12S rRNA is the primary target for aminoglycoside-induced deafness. These results also strongly suggest that the nuclear background plays a role in the aminoglycoside ototoxicity and in the development of the deafness phenotype associated with the C1494T mutation in the mitochondrial 12S rRNA gene.     

Mitochondrial haplotypes may modulate the phenotypic manifestation of the deafness-associated 12S rRNA 1555A>G mutation

Mitochondrial 12S rRNA 1555A>G mutation is one of the important causes of aminoglycoside-induced and nonsyndromic deafness. Our previous investigations showed that the A1555G mutation was a primary factor underlying the development of deafness but was insufficient to produce deafness phenotype. However, it has been proposed that mitochondrial haplotypes modulate the phenotypic manifestation of the 1555A>G mutation. Here, we performed systematic and extended mutational screening of 12S rRNA gene in a cohort of 1742 hearing-impaired Han Chinese pediatric subjects from Zhejiang Province, China. Among these, 69 subjects with aminoglycoside-induced and nonsyndromic deafness harbored the homoplasmic 1555A>G mutation. These translated to a frequency of ～3.96% for the 1555A>G mutation in this hearing–impaired population. Clinical and genetic characterizations of 69 Chinese families carrying the 1555A>G mutation exhibited a wide range of penetrance and expressivity of hearing impairment. The average penetrances of deafness were 29.5% and 17.6%, respectively, when aminoglycoside-induced hearing loss was included or excluded. Furthermore, the average age-of-onset for deafness without aminoglycoside exposure ranged from 5 and 30 years old, with the average of 14.5 years. Their mitochondrial genomes exhibited distinct sets of polymorphisms belonging to ten Eastern Asian haplogroups A, B, C, D, F, G, M, N, R and Y, respectively. These indicated that the 1555A>G mutation occurred through recurrent origins and founder events. The haplogroup D accounted for 40.6% of the patient’s mtDNA samples but only 25.8% of the Chinese control mtDNA samples. Strikingly, these Chinese families carrying mitochondrial haplogroup B exhibited higher penetrance and expressivity of hearing loss. In addition, the mitochondrial haplogroup specific variants: 15927G>A of haplogroup B5b, 12338T>C of haplogroup F2, 7444G>A of haplogroup B4, 5802T>C, 10454T>C, 12224C>T and 11696G>A of D4 haplogroup, 5821G>A of haplogroup C, 14693A>G of haplogroups Y2 and F, and 15908T>C of Y2 may enhance the penetrace of hearing loss in these Chinese families. Moreover, the absence of mutation in nuclear modifier gene TRMU suggested that TRMU may not be a modifier for the phenotypic expression of the 1555A>G mutation in these Chinese families. These observations suggested that mitochondrial haplotypes modulate the variable penetrance and expressivity of deafness among these Chinese families.     

Biogenesis of mitochondria 51

Summary An examination of the effect of the aminoglycoside antibiotics paromomycin and neomycin on mitochondrial ribosome function in yeast has been made. Both antibiotics are potent inhibitors of protein synthesis in isolated mitochondria. With isolated mitochondrial ribosomes programmed with polyuridylic acid (poly U), the drugs are shown to inhibit polyphenylalanine synthesis at moderately high concentrations (above 100 g/ml). At lower concentrations (about 10 g/ml), paromomycin and neomycin cause a 2–3 fold stimulation in the extent of misreading of the UUU codons in poly U, over and above the significant level of misreading catalyzed by the ribosomes in the absence of drugs.Comparative studies have been made between a paromomycin sensitive strain D585-11C and a mutant strain 4810P carrying the parl-r mutation in mtDNA, which leads tohigh resistance to both paromomycin and neomycin in vivo. A high level of resistance to these antibiotics is observed in strain 4810P at the level of mitochondrial protein synthesis in vitro. Whilst the degree of resistance of isolated mitochondrial ribosomes from strain 4810P judged by the inhibition of polyphenylalanine synthesis by paromomycin and neomycin is not extensive, studies on misreading of the poly U message promoted by these drugs demonstrate convincingly the altered properties of mitochondrial ribosomes from the mutant strain 4810P. These ribosomes show resistance to the stimulation of misreading of the codon UUU brought about by paromomycin and neomycin in wild-type mitochondrial ribosomes. Although strain 4810P was originally isolated as being resistant to paromomycin, in all the in vitro amino acid incorporation systems tested here, the 4810P mitochondrial ribosomes show a higher degree of resistance to neomycin than to paromomycin.It is concluded that the parl-r mutation in strain 4810P affects a component of the mitochondrial ribosome, possibly by altering the 15S rRNA or a protein of the small ribosomal subunit. The further elucidation of the functions in the ribosomes that are modified by the parl-r mutation was hampered by the inability of current preparations of yeast mitochondrial ribosomes to translate efficiently natural messenger RNAs from the several sources tested.     

The yeast counterparts of human 'MELAS' mutations cause mitochondrial dysfunction that can be rescued by overexpression of the mitochondrial translation factor EF-Tu

We have taken advantage of the similarity between human and yeast (Saccharomyces cerevisiae) mitochondrial tRNA^Leu(UUR), and of the possibility of transforming yeast mitochondria, to construct yeast mitochondrial mutations in the gene encoding tRNA^Leu(UUR) equivalent to the human A3243G, C3256T and T3291C mutations that have been found in patients with the neurodegenerative disease MELAS (for mitochondrial 'myopathy, encephalopathy, lactic acidosis and stroke-like episodes'). The resulting yeast cells (bearing the equivalent mutations A14G, C26T and T69C) were defective for growth on respiratory substrates, exhibited an abnormal mitochondrial morphology, and accumulated mitochondrial DNA deletions at a very high rate, a trait characteristic of severe mitochondrial defects in protein synthesis. This effect was specific at least in the pathogenic mutation T69C, because when we introduced A or G instead of C, the respiratory defect was absent or very mild. All defective phenotypes returned to normal when the mutant cells were transformed by multicopy plasmids carrying the gene encoding the mitochondrial elongation factor EF-Tu. The ability to create and analyse such mutated strains and to select correcting genes should make yeast a good model for the study of tRNAs and their interacting partners and a practical tool for the study of pathological mutations and of tRNA sequence polymorphisms.     

Substitution of an invariant nucleotide at the base of the highly conserved ''530-loop'' of 15S rRNA causes suppression of yeast mitochondrial ochre mutations.

We have determined the nucleotide sequence alteration in the 15S rRNA gene of a Saccharomyces cerevisiae strain carrying the previously described mitochondrial ochre suppressor, MSUI. The suppressor contains an A residue at position 633 of the yeast mitochondrial sequence, in place of the wild-type G. This position, located in the highly conserved region forming the stem of the '530-loop', corresponds to G517 of the Escherichia coli 16S rRNA and is occupied by G in all other known small rRNA sequences. This finding strongly supports the previous conclusions of others that the 530-loop region plays an important role in enhancing translational accuracy.     

Identification and characterization of mouse MTO1 gene related to mitochondrial tRNA modification

The nucleotide modification in tRNA plays a pivotal role in the fidelity of translational process. The defects in nucleotide modification have often been observed in the mutated mitochondrial tRNAs associated with human diseases. Recently, MTO1-like protein in bacteria and yeast has been implicated to be a component of tRNA modification pathway. Here we report the identification and characterization of mouse MTO1 homolog. The mouse MTO1 gene containing 12 exons encodes a 669-residue protein with a strong homology to the MTO1-like proteins of bacteria and yeast, related to tRNA modification. Functional conservation of this protein is supported by the observation that the isolated mouse MTO1 cDNA can complement the respiratory-deficient phenotype of yeast mto1 cells carrying P(R)(454) mutation. MTO1 is ubiquitously expressed in various tissues, but with markedly elevated expression in tissues of high metabolic rates. Furthermore, we showed that mouse Mto1 localizes in mitochondrion. These observations suggest that the mouse MTO1 is a structural and functional homolog of yeast MTO1, thereby playing a role in the mitochondrial tRNA modification and protein synthesis.