人线粒体tRNALeu(UUR)基因A3243G点突变对其亮氨酰化活性的影响

化学法合成人线粒体野生型与A3243G点突变型tRNALeu(UUR)基因,体外转录生成相应的tRNALeu(UUR),表达并纯化人线粒体亮氨酰tRNA合成酶(mtLeuRS),用mtLeuRS催化野生型与突变型tRNALeu(UUR)与亮氨酸结合,分别检测两种类型tRNALeu(UUR)的氨酰化动力学常数。结果表明,野生型tRNALeu(UUR)的Km/Kcat仅为突变型tRNALeu(UUR)的63.9%,A3243G点突变使tRNALeu(UUR)接受亮氨酸的能力明显下降,提示此为A3243G点突变致病机制之一。 Abstract:The wild-type and mutant-type human mitochondrial tRNALeu(UUR) genes were synthesized and transcribed in vitro with T7 RNA polymerase.The kinetic parameters of human mitochondrial leucyl-tRNA synthetase(mtLeuRS) were determined with wild-type and mutant-type human mitochondrial tRNALeu(UUR) respectively.The results show that the value of Km/Kcat of mtLeuRS for the mutant-type tRNALeu(UUR) is 63.9% as compared with the wild-type.Human mitochondrial tRNALeu(UUR) gene A3243G point mutant can remarkably reduce it′s aminoacylation activity,suggesting it would be one of the mechanisms that the mutation could produce such clinical phenotypes.     

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.     

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.     

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.     

Mutational analysis of the mitochondrial tRNALeu(UUR) gene in Tunisian patients with mitochondrial diseases

The mitochondrial tRNA(Leu(UUR)) gene (MTTL) is a hot spot for pathogenic mutations that are associated with mitochondrial diseases with various clinical features. Among these mutations, the A3243G mutation was associated with various types of mitochondrial multisystem disorders, such as MIDD, MELAS, MERRF, PEO, hypertrophic cardiomyopathy, and a subtype of Leigh syndrome. We screened 128 Tunisian patients for the A3243G mutation in the mitochondrial tRNA(Leu(UUR)) gene. This screening was carried out using PCR-RFLP with the restriction endonuclease ApaI. None of the 128 patients or the 100 controls tested were found to carry the mitochondrial A3243G mutation in the tRNA(Leu(UUR)) gene in homoplasmic or heteroplasmic form. After direct sequencing of the entire mitochondrial tRNA(Leu(UUR)) gene and a part of the mitochondrial NADH dehydrogenase 1, we found neither mutations nor polymorphisms in the MTTL1 gene in the tested patients and controls, and we confirmed the absence of the A3243G mutation in this gene. We also found a T3396C transition in the ND1 gene in one family with NSHL which was absent in the other patients and in 100 controls. Neither polymorphisms nor other mutations were found in the mitochondrial tRNA(Leu(UUR)) gene in the tested patients.     

Interdomain communication between weak structural elements within a disease-related human tRNA

The structure of the human mitochondrial (hs mt) tRNALeu(UUR) features several domains that are predicted to exhibit limited thermodynamic stability. An elevated frequency of disease-related mutations within these domains suggests a link between structural instability and the functional effects of pathogenic mutations. A series of tRNAs featuring mutations within the D and anticodon stems were prepared and investigated using nuclease probing. Structural mapping studies indicated that these domains were partially denatured for the wild type (WT) hs mt tRNALeu(UUR) and were significantly stabilized by mutations introducing additional or stronger base pairs into the stem regions. In addition, trends in the aminoacylation activities of the D stem mutants suggested that the loose structure is required for function, with mutants displaying the most ordered structures exhibiting the lowest levels of aminoacylation activity. A pronounced interdependence of the structures of the anticodon and D stems was observed, with mutations strengthening the D stem stabilizing the anticodon stem and vice versa. The existence of strong interdomain communication was further elucidated with a mutant of hs mt tRNALeu(UUR) containing a stabilized D stem and a pathogenic mutation that disrupted the anticodon stem. Strengthening the structure of the D stem completely restored the function of the disease-related mutant to WT levels, indicating that propagated structural weaknesses contribute to the functional deactivation of this tRNA by mutations.     

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.     

Abnormal cardiac energetics in patients carrying the A3243G mtDNA mutation measured in vivo using phosphorus MR spectroscopy

Cardiomyopathy is a frequent cause of morbidity and mortality in patients carrying the A3243G transition in the mitochondrial DNA (mtDNA) tRNALeu(UUR) gene, the most common heteroplasmic single mtDNA defect. We used phosphorus magnetic resonance spectroscopy (31P-MRS) to look for evidence of an in vivo bioenergetics defect in patients carrying the A3243G mtDNA mutation with and without echocardiographic signs of left ventricle hypertrophy (LVH). Eight patients, three with LVH, carrying the A3243G mtDNA mutation and 10 healthy subjects underwent one-dimensional chemical shift imaging 31P-MRS. In the patients, mean cardiac phosphocreatine to adenosine triphosphate ratio (PCr/ATP) (1.55 +/- 0.58) was significantly reduced compared to the control group (2.34 +/- 0.14; P < 0.001). Cardiac PCr/ATP was within the normal range only in one case that showed normal echocardiography. Our results point to a central role of bioenergetics deficit in the development of cardiac hypertrophy in patients with the A3243G mtDNA mutation. Impaired cardiac energy metabolism in patients with normal echocardiography suggests that the enhancement of mitochondrial function may be beneficial not only to patients with cardiac hypertrophy but also to those patients carrying the mutation in the absence of signs of cardiac hypertrophy and/or dysfunction but with cardiac bioenergetics deficit.     

The pathogenic U3271C human mitochondrial tRNA(Leu(UUR)) mutation disrupts a fragile anticodon stem

The U3271C mutation affecting the human mitochondrial transfer RNA(Leu(UUR)) (hs mt tRNA) is correlated with diabetes and mitochondrial encephalopathies. We have explored the relationship between the structural effects of this mutation and its impact on function using chemical probing experiments and in vitro aminoacylation assays to investigate a series of tRNA constructs. Chemical probing experiments indicate that the U3271C substitution, which replaces an AU pair with a CA mispair, significantly destabilizes the anticodon stem. The introduction of a compensatory A3261G mutation reintroduces base pairing at this site and restores the structure of this domain. In fact, the anticodon stem of the A3261G/U3271C mutant appears more structured than wild-type (WT) hs mt tRNA(Leu(UUR)), indicating that the entirely AU stem of the native tRNA is intrinsically weak. The results of the chemical probing experiments are mirrored in the aminoacylation activities of the mutants. The U3271C substitution decreases aminoacylation reactivity relative to the WT tRNA due to an increase in K(m) for the pathogenic mutant. The binding defect is a direct result of the structural disruption caused by the pathogenic mutation, as the introduction of the stabilizing compensatory mutation restores aminoacylation activity. Other examples of functional defects associated with the disruption of weak domains in hs mt tRNAs have been reported, indicating that the effects of pathogenic mutations may be amplified by the fragile structures that are characteristic of this class of tRNAs.     

人线粒体tRNA修饰碱基与遗传性脑肌病

人的多种遗传疾病与线粒体tRNA基因突变有关,这些突变导致疾病发生的分子机理是当前研究的热点.通过研究线粒体tRNA分子上的碱基修饰情况,人们发现了一类特殊的带有牛磺酸衍生物基团的修饰,这类修饰主要位于线粒体tRNALys和线粒体tRNALeu(UUR)反密码子第一位摆动(wobble)位点的碱基上.最近的研究表明,位于这两种线粒体tRNA基因上的多种突变与遗传性脑肌病相关,包括A8344G,A3243G,T3271C等等,它们可以导致tRNA上相应摆动位点的碱基修饰缺失.无论是在体外培养的带有相应突变的细胞内,还是在来源于脑肌病病人的组织中,科学家都发现了相同的线粒体tRNA碱基修饰缺陷.通过分子手术证实,此类碱基修饰对于维持这两种tRNA的反密码子与mRNA上相应密码子的相互识别至关重要,缺失了这种修饰的tRNA将无法识别一些对应的密码子.通过进一步的实验,人们还鉴定出负责催化此类碱基修饰的酶.这些研究不但揭示了线粒体遗传性脑肌病相关突变的致病机理,也将为研究基因治疗提供可能的新手段.     

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.