Dimerization of a pathogenic human mitochondrial tRNA

Mutations of human mitochondrial transfer RNA (tRNA) are implicated in a variety of multisystemic diseases. The most prevalent pathogenic mitochondrial mutation is the A3243G substitution within the gene for tRNA(Leu(UUR)). Here we describe the pronounced structural change promoted by this mutation. The A3243G mutation induces the formation of a tRNA dimer that strongly self-associates under physiological conditions. The dimerization interface in the mutant tRNA is a self-complementary hexanucleotide in the D-stem, a particularly weak structural element within tRNA(Leu(UUR)). Aminoacylation of the A3243G mutant is significantly attenuated, and mutational studies indicate that dimerization is partially responsible for the observed loss of function. The disruption of a conserved tertiary structural contact also contributes to the functional defect. The pathogenic mutation is proposed to interfere with the cellular function of human mitochondrial tRNA(Leu(UUR)) by destabilizing the native structure and facilitating the formation of a dimeric complex with low biological activity.     

Overexpressed mitochondrial leucyl-tRNA synthetase suppresses the A3243G mutation in the mitochondrial tRNA(Leu(UUR)) gene

The A3243G mutation in the human mitochondrial tRNA^Leu(UUR) gene causes a number of human diseases. This mutation reduces the level and fraction of aminoacylated tRNA^Leu(UUR) and eliminates nucleotide modification at the wobble position of the anticodon. These deficiencies are associated with mitochondrial translation defects that result in decreased levels of mitochondrial translation products and respiratory chain enzyme activities. We have suppressed the respiratory chain defects in A3243G mutant cells by overexpressing human mitochondrial leucyl-tRNA synthetase. The rates of oxygen consumption in suppressed cells were directly proportional to the levels of leucyl-tRNA synthetase. Fifteenfold higher levels of leucyl-tRNA synthetase resulted in wild-type respiratory chain function. The suppressed cells had increased steady-state levels of tRNA^Leu(UUR) and up to threefold higher steady-state levels of mitochondrial translation products, but did not have rates of protein synthesis above those in parental mutant cells. These data suggest that suppression of the A3243G mutation occurred by increasing protein stability. This suppression of a tRNA gene mutation by increasing the steady-state levels of its cognate aminoacyl-tRNA synthetase is a model for potential therapies for human pathogenic tRNA mutations.     

Modification defect at anticodon wobble nucleotide of mitochondrial tRNAs(Leu)(UUR) with pathogenic mutations of mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes

The mitochondrial tRNA(Leu)(UUR) (R = A or G) gene possesses several hot spots for pathogenic mutations. A point mutation at nucleotide position 3243 or 3271 is associated with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes and maternally inherited diabetes with deafness. Detailed studies on two tRNAs(Leu)(UUR) with the 3243 or 3271 mutation revealed some common characteristics in cybrid cells: (i) a decreased life span, resulting in a 70% decrease in the amounts of the tRNAs in the steady state, (ii) a slight decrease in the ratios of aminoacyl-tRNAs(Leu)(UUR) versus uncharged tRNAs(Leu)(UUR), and (iii) accurate aminoacylation with leucine without any misacylation. As a marked result, both of the mutant tRNA molecules were deficient in a modification of uridine that occurs in the normal tRNA(Leu)(UUR) at the first position of the anticodon. The lack of this modification may lead to the mistranslation of leucine into non-cognate phenylalanine codons by mutant tRNAs(Leu)(UUR), according to the mitochondrial wobble rule, and/or a decrease in the rate of mitochondrial protein synthesis. This finding could explain why two different mutations (3243 and 3271) manifest indistinguishable clinical features.     

Correction of the consequences of mitochondrial 3243A>G mutation in the MT-TL1 gene causing the MELAS syndrome by tRNA import into mitochondria

Mutations in human mitochondrial DNA are often associated with incurable human neuromuscular diseases. Among these mutations, an important number have been identified in tRNA genes, including 29 in the gene MT-TL1 coding for the tRNA(Leu(UUR)). The m.3243A>G mutation was described as the major cause of the MELAS syndrome (mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes). This mutation was reported to reduce tRNA(Leu(UUR)) aminoacylation and modification of its anti-codon wobble position, which results in a defective mitochondrial protein synthesis and reduced activities of respiratory chain complexes. In the present study, we have tested whether the mitochondrial targeting of recombinant tRNAs bearing the identity elements for human mitochondrial leucyl-tRNA synthetase can rescue the phenotype caused by MELAS mutation in human transmitochondrial cybrid cells. We demonstrate that nuclear expression and mitochondrial targeting of specifically designed transgenic tRNAs results in an improvement of mitochondrial translation, increased levels of mitochondrial DNA-encoded respiratory complexes subunits, and significant rescue of respiration. These findings prove the possibility to direct tRNAs with changed aminoacylation specificities into mitochondria, thus extending the potential therapeutic strategy of allotopic expression to address mitochondrial disorders.     

Wobble modification deficiency in mutant tRNAs in patients with mitochondrial diseases

Point mutations in mitochondrial (mt) tRNA genes are associated with a variety of human mitochondrial diseases. We have shown previously that mt tRNA(Leu(UUR)) with a MELAS A3243G mutation and mt tRNA(Lys) with a MERRF A8344G mutation derived from HeLa background cybrid cells are deficient in normal taurine-containing modifications [taum(5)(s(2))U; 5-taurinomethyl-(2-thio)uridine] at the anticodon wobble position in both cases. The wobble modification deficiency results in defective translation. We report here wobble modification deficiencies of mutant mt tRNAs from cybrid cells with different nuclear backgrounds, as well as from patient tissues. These findings demonstrate the generality of the wobble modification deficiency in mutant tRNAs in MELAS and MERRF.     

温州地区2型糖尿病患者线粒体DNA 3243、3316位点的突变筛查

为了解浙江省温州地区2型糖尿病病人中线粒体DNA tRNALeu (UUR)基因A3243G及NADH 脱氢酶亚单位1 (ND1)基因G3316A位点突变的发生频率, 并探讨突变与2型糖尿病主要临床指标出现的相关性。对随机收集的无血缘关系的244例温州地区2型糖尿病患者进行研究, 同时选择156例无 DM 家族史的糖耐量正常者作为对照组, 用聚合酶链反应及限制性片段长度多态性分析技术进行点突变筛选, 筛选到的异质性突变样本经T-A克隆后再作测序和变性高效液相色谱(DHPLC)确证。结果在244例的2型糖尿病患者中检出A3243G突变1例(0.410%), 156例对照者中未检出该突变, 突变发生率在两组间差异无统计学意义(P>0.05); 2型糖尿病患者中检出G3316A突变4例(1.639%), 156例对照者中检出突变2例(1. 282%), 突变发生率在两组间差异无统计学意义(P>0.05)。结果表明线粒 体tRNALeu (UUR) 基因A3243G突变在浙江温州2型糖尿病人群中发生频率低, 不是温州人群中2型糖尿病的常见病因。线粒体ND1基因G3316A突变在糖尿病人群中的发生频率也较低, 且在正常人群中也有出现, 可能仅为人群中线粒体DNA的基因多态性。     

Maternally transmitted diabetes mellitus associated with the mitochondrial tRNA(Leu(UUR)) A3243G mutation in a four-generation Han Chinese family

We report here the characterization of a four-generation Han Chinese family with maternally transmitted diabetes mellitus. Six (two males/four females) of eight matrilineal relatives in this family exhibited diabetes. The age of onset in diabetes varies from 15 years to 33 years, with an average of 26 years. Two of affected matrilineal relatives also exhibited hearing impairment. Molecular analysis of mitochondrial DNA (mtDNA) showed the presence of heteroplasmic tRNA(Lue(UUR)) A3243G mutation, ranging from 35% to 58% of mutations in blood cells of matrilineal relatives. The levels of heteroplasmic A3243G mutation seem to be correlated with the severity and age-at-onset of diabetes in this family. Sequence analysis of the complete mitochondrial genome in this pedigree revealed the presence of the A3243G mutation and 38 other variants belonging to the Eastern Asian haplogroup M7C. However, none of other mtDNA variants are evolutionarily conserved and implicated to have significantly functional consequence. Thus, the A3243G mutation is the sole pathogenic mtDNA mutation associated with diabetes in this Chinese family.     

A new mtDNA mutation associated with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS)

In 3 of 40 MELAS patients, a new common mutation, a T-to-C transition at nucleotide position 3271 in the mitochondrial tRNA(Leu(UUR] gene was recognized and was very near to the most common mutation site at 3243. With a simple detection method using polymerase chain reaction with a mismatch primer, none of 46 patients with other mitochondrial diseases and 50 controls had this mutation.     

Mutations in mitochondrial tRNA genes: non-linkage with syndromes of Wolfram and chronic progressive external ophthalmoplegia.

We have recently identified a point mutation in the mitochondrially encoded tRNA(Leu(UUR)) gene which associates with a combination of type II diabetes mellitus and sensorineural hearing loss in a large pedigree. To extend this finding to other syndromes which exhibit a combination of diabetes mellitus and hearing loss we have sequenced all mitochondrial tRNA genes from two patients with the Wolfram syndrome, a rare congenital disease characterized by diabetes mellitus, deafness, diabetes insipidus and optic atrophy. In each patient, a single different mutation was identified. One is an A to G transition mutation at np 12,308 in tRNA(Leu(CUN)) gene in a region which is highly conserved between species during evolution. This mutation has been described by Lauber et al. (1) as associating with chronic progressive external ophthalmoplegia (CPEO). The other is a C to T transition mutation at np 15,904 in tRNA(Thr) gene. Both mutations are also present in the general population (frequency tRNA(Leu(CUN)) mutation 0.16, tRNA(Thr) mutation 0.015). These findings suggest that evolutionarily conserved regions in mitochondrial tRNA genes can exhibit a significant polymorphism in humans, and that the mutation at np 12,308 in the tRNA(Leu(CUN)) gene is unlikely to be associated with CPEO and Wolfram syndrome.     

Mitochondrial DNA mutations in mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS)

The total sequences of mitochondrial DNA were determined in two patients with juvenile-onset mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) due to Complex I deficiency. Patients 1 and 2 had three and two unique point mutations, respectively, causing replacement of phylogenically conserved amino acids. A transition from G to A was found at nucleotide position 5601 in the alanine tRNA gene of Patient 2, and a transition from A to G was found at 3243 in the leucine (UUR) tRNA gene of both patients. The latter mutation located at the phylogenically conserved 5' end of the dihydrouridine loop of the tRNA molecule, and was present in two patients with adult-onset MELAS and absent in controls. These results indicate that a mass of mtDNA mutations including the A-to-G transition in the tRNA(Leu) gene is a genetic cause of MELAS.     

Mitochondrial tRNA mutations associated with deafness

Mitochondrial tRNA mutations are one of the important causes of both syndromic and non-syndromic deafness. Of those, syndromic deafness-associated tRNA mutations such as tRNA(Leu(UUR)) 3243A>G are often present in heteroplasmy, while non-syndromic deafness-associated tRNA mutations including tRNA(Ser(UCN)) 7445A>G often occur in homplasmy or in high levels of heteroplasmy. These tRNA mutations are the primary mutations leading to hearing loss. However, other tRNA mutations such as tRNA(Thr) 15927G>A and tRNA(Ser(UCN)) 7444G>A may act in synergy with the primary mitochondrial DNA mutations, modulating the phenotypic manifestation of the primary mitochondrial DNA mutations. Theses tRNA mutations cause structural and functional alteration. A failure in tRNA metabolism caused by these tRNA mutations impaired mitochondrial translation and respiration, thereby causing mitochondrial dysfunctions responsible for deafness. These data offer valuable information for the early diagnosis, management and treatment of maternally inherited deafness.     

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.     

The diabetes-associated 3243 mutation in the mitochondrial tRNA(Leu(UUR)) gene causes severe mitochondrial dysfunction without a strong decrease in protein synthesis rate.

Cells harboring patient-derived mitochondria with an A-to-G transition at nucleotide position 3243 of their mitochondrial DNA display severe loss of respiration when compared with cells containing the wild-type adenine but otherwise identical mitochondrial DNA sequence. The amount and degree of leucylation of tRNA(Leu(UUR)) were both found to be highly reduced in mutant cells. Despite the low level of leucyl-tRNA(Leu(UUR)), the rate of mitochondrial translation was not seriously affected by this mutation. Therefore, decrease of mitochondrial protein synthesis as such does not appear to be a necessary prerequisite for loss of respiration. Rather, the mitochondrially encoded proteins seem subject to elevated degradation, leading to a severe reduction in their steady state levels. Our results favor a scheme in which the 3243 mutation causes loss of respiration through accelerated protein degradation, leading to a disequilibrium between the levels of mitochondrial and nuclear encoded respiratory chain subunits and thereby a reduction of functional respiratory chain complexes. The possible mechanisms underlying the pathogenesis of mitochondrial diabetes is discussed.     

Yeast as a model of human mitochondrial tRNA base substitutions: investigation of the molecular basis of respiratory defects

We investigate the relationships between acylation defects and structure alterations due to base substitutions in yeast mitochondrial (mt) tRNA(UUR)(Leu). The studied substitutions are equivalent to the A3243G and T3250C human pathogenetic tRNA mutations. Our data show that both mutations can produce tRNA(UUR)(Leu) acylation defects, although to a different extent. For mutant A14G (equivalent to MELAS A3243G base substitution), the presence of the tRNA and its defective aminoacylation could be observed only in the nuclear context of W303, a strain where the protein synthesis defects caused by tRNA base substitutions are far less severe than in previously studied strains. For mutant T20C (equivalent to the MM/CPEO human T3250C mutation), the acylation defect was less severe, and a thermosensitive acylation could be detected also in the MCC123 strain. The correlation between the severity of the in vivo phenotypes of yeast tRNA mutants and those obtained in in vitro studies of human tRNA mutants supports the view that yeast is a suitable model to study the cellular and molecular effects of tRNA mutations involved in human pathologies. Furthermore, the yeast model offers the possibility of modulating the severity of yeast respiratory phenotypes by studying the tRNA mutants in different nuclear contexts. The nucleotides at positions 14 and 20 are both highly conserved in yeast and human mt tRNAs; however, the different effect of their mutations can be explained by structure analyses and quantum mechanics calculations that can shed light on the molecular mechanisms responsible for the experimentally determined defects of the mutants.