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.     

Mitochondrial leucine tRNA level and PTCD1 are regulated in response to leucine starvation

Pentatricopeptide repeat domain protein 1 (PTCD1) is a novel human protein that was recently shown to decrease the levels of mitochondrial leucine tRNAs. The physiological role of this regulation, however, remains unclear. Here we show that amino acid starvation by leucine deprivation significantly increased the mRNA steady-state levels of PTCD1 in human hepatocarcinoma (HepG2) cells. Amino acid starvation also increased the mitochondrially encoded leucine tRNA (tRNA^Leu(CUN)) and the mRNA for the mitochondrial leucyl-tRNA synthetase (LARS2). Despite increased PTCD1 mRNA steady-state levels, amino acid starvation decreased PTCD1 on the protein level. Decreasing PTCD1 protein concentration increases the stability of the mitochondrial leucine tRNAs, tRNA^Leu(CUN) and tRNA^Leu(UUR) as could be shown by RNAi experiments against PTCD1. Therefore, it is likely that decreased PTCD1 protein contributes to the increased tRNA^Leu(CUN) levels in amino acid-starved cells. The stabilisation of the mitochondrial leucine tRNAs and the upregulation of the mitochondrial leucyl-tRNA synthetase LARS2 might play a role in adaptation of mitochondria to amino acid starvation.     

Chemical synthesis of the 5-taurinomethyl(-2-thio)uridine modified anticodon arm of the human mitochondrial tRNALeu(UUR) and tRNALys

5-Taurinomethyluridine (τm⁵U) and 5-taurinomethyl-2-thiouridine (τm⁵s²U) are located at the wobble position of human mitochondrial (hmt) tRNA^Leu(UUR) and tRNA^Lys, respectively. Both hypermodified units restrict decoding of the third codon letter to A and G. Pathogenic mutations in the genes encoding hmt-tRNA^Leu(UUR) and hmt-tRNA^Lys are responsible for the loss of the discussed modifications and, as a consequence, for the occurrence of severe mitochondrial dysfunctions (MELAS, MERRF). Synthetic oligoribonucleotides bearing modified nucleosides are a versatile tool for studying mechanisms of genetic message translation and accompanying pathologies at nucleoside resolution. In this paper, we present site-specific chemical incorporation of τm⁵U and τm⁵s²U into 17-mers related to the sequence of the anticodon arms hmt-tRNA^Leu(UUR) and hmt-tRNA^Lys, respectively employing phosphoramidite chemistry on CPG support. Selected protecting groups for the sulfonic acid (4-(tert-butyldiphenylsilanyloxy)-2,2-dimethylbutyl) and the exoamine function (-C(O)CF₃) are compatible with the blockage of the canonical monomeric units. The synthesis of τm⁵s²U-modified RNA fragment was performed under conditions eliminating the formation of side products of 2-thiocarbonyl group oxidation and/or oxidative desulphurization. The structure of the final oligomers was confirmed by mass spectroscopy and enzymatic cleavage data.     

与耳聋相关的线粒体tRNA突变

线粒体tRNA基因突变是导致感音神经性耳聋的原因之一．有些tRNA突变可直接造成耳聋的发生,称之为原发突变．如tRNA^Leu(UUR) A3243G等突变与综合征型耳聋相关,而tRNA^Ser(UCN) T7511C等突变则与非综合征型耳聋相关．此外,继发突变如tRNA^Thr G15927A等突变则对原发突变起协同作用,影响耳聋的表型表达．这些突变可引起tRNA二级结构改变,从而影响线粒体蛋白质合成,降低细胞内ATP的产生,由此引起的线粒体功能障碍可导致耳聋的发生．主要讨论与耳聋相关的线粒体tRNA突变及其致聋机理．     

Gene cloning, expression and purification of human mitochondrial tRNALeu(UUR) and its mutant

Genes of human mitochondrial tRNA^Leu(UUR) (mtRNA^Leu(UUR)) and its mutant (mtRNA^Leu(M)) were synthesized and inserted into the plasmid pGEM-9Zf(-) respectively.E.coli JM 109 was transformed by the recombinant plasmids containing the target genes. The mtRNA^Leu(UUR) and mtRNA^Leu(M) were expressed up to 19.10% and 17.76% of total small RNA respectively. They were purified to 54% homogeneity by DEAE-sepharose-CL4B column chromatography and finally repurified by 15% PAGE/urea. Their kinetic parameters forE.coli LeuRS were measured. The results showed that the value of k_cal/ K_m of mtRNA^Leu(M) was about one fifth of that of mtRNA^Leu(UUR) and indicated the leucine acceptability of mtRNA^Leu(M) was much lower than that of mtRNA^Leu(UUR).     

Gene cloning, expression and purification of human mitochondrial tRNALeu(UUR) and its mutant

Genes of human mitochondrial tRNA^Leu(UUR) (mtRNA^Leu(UUR)) and its mutant (mtRNA^Leu(M)) were synthesized and inserted into the plasmid pGEM-9Zf(-) respectively.E.coli JM 109 was transformed by the recombinant plasmids containing the target genes. The mtRNA^Leu(UUR) and mtRNA^Leu(M) were expressed up to 19.10% and 17.76% of total small RNA respectively. They were purified to 54% homogeneity by DEAE-sepharose-CL4B column chromatography and finally repurified by 15% PAGE/urea. Their kinetic parameters forE.coli LeuRS were measured. The results showed that the value of k_cal/ K_m of mtRNA^Leu(M) was about one fifth of that of mtRNA^Leu(UUR) and indicated the leucine acceptability of mtRNA^Leu(M) was much lower than that of mtRNA^Leu(UUR).     

Multiple mitochondrial tRNAMLeu[UUR] mutations associated with infantile myopathy

We have identified a cluster of mitochondrial tRNA^Leu[UUR], mutations in a severe case of infantile myopathy. There were A to G transitions found at mtDNA positions 3259, 3261, 3266 and 3268. These point mutations change the anticodon arm and the anticodon UAA, normally found in tRNA^Leu[UUR], to UGA which is the one of the tRNAs^Ser[UCN]. This is the first anticodon alteration described in this tRNA. Another swap straight to the anticodon of tRNA^Pro alone was recently described in a less severe case [1]. Until now infantile myopathies have not been attributed to defined mtDNA alterations. This study reports for the first time mtDNA point mutations causing this early onset of a mitochondrial disorder. The apparent homoplasmy of these mutations and especially the location in the anticodon must be considered lethal, if the child would not have been respirated for 5 years from its birth. (Mol Cell Biochem 174: 231–236, 1997)     

Molecular pathology of MELAS and l-arginine effects

Background

The pathogenic mechanism of stroke-like episodes seen in mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) has not been clarified yet. About 80% of MELAS patients have an A3243G mutation in the mitochondrial tRNA^Leu(UUR) gene, which is the base change at position 14 in the consensus structure of tRNA^Leu(UUR) gene.

Scope of review

This review aims to give an overview on the actual knowledge about the pathogenic mechanism of mitochondrial cytopathy at the molecular levels, the possible pathogenic mechanism of mitochondrial angiopathy to cause stroke-like episodes at the clinical and pathophysiological levels, and the proposed site of action of l-arginine therapy on MELAS.

Major conclusions

Molecular pathogenesis is mainly demonstrated using ρ⁰ cybrid system. The mutation creates the protein synthesis defects caused by 1) decreased life span of steady state amount of tRNA^Leu(UUR) molecules; 2) decreased ratio of aminoacyl-tRNA^Leu(UUR) versus uncharged tRNA^Leu(UUR) molecules; 3) the accumulation of aminoacylation with leucine without any misacylation; 4) accumulation of processing intermediates such as RNA 19, 5) wobble modification defects. All of these loss of function abnormalities are created by the threshold effects of cell or organ to the mitochondrial energy requirement when they establish the phenotype. Mitochondrial angiopathy demonstrated by muscle or brain pathology, as SSV (SDH strongly stained vessels), and by vascular physiology using FMD (flow mediated dilation). MELAS patients show decreased capacity of NO dependent vasodilation because of the low plasma levels of l-arginine and/or of respiratory chain dysfunction. Although the underlying mechanisms are not completely understood in stroke-like episodes in MELAS, l-arginine therapy improved endothelial dysfunction.

General significance

Though the molecular pathogenesis of an A3243G or T3271C mutation of mitochondrial tRNA^Leu(UUR) gene has been clarified as a mitochondrial cytopathy, the underlying mechanisms of stroke-like episodes in MELAS are not completely understood. At this point, l-arginine therapy showed promise in treating of the stroke-like episodes in MELAS. This article is part of a Special Issue entitled Biochemistry of Mitochondria.     

Biochemical characterization of the mitochondrial tRNASer(UCN) T7511C mutation associated with nonsyndromic deafness

We report here the biochemical characterization of the deafness-associated mitochondrial tRNA^Ser(UCN) T7511C mutation, in conjunction with homoplasmic ND1 T3308C and tRNA^Ala T5655C mutations using cybrids constructed by transferring mitochondria from lymphoblastoid cell lines derived from an African family into human mtDNA-less (ρ°) cells. Three cybrids derived from an affected matrilineal relative carrying the homoplasmic T7511C mutation, exhibited ~75% decrease in the tRNA^Ser(UCN) level, compared with three control cybrids. This amount of reduction in the tRNA^Ser(UCN) level is below a proposed threshold to support a normal rate of mitochondrial protein synthesis in lymphoblastoid cell lines. This defect is likely a primary contributor to ~52% reduction in the rate of mitochondrial protein synthesis and marked defects in respiration and growth properties in galactose-containing medium. Interestingly, the T5655C mutation produces ~50% reduction in the tRNA^Ala level in mutant cells. Strikingly, the T3308C mutation causes a significant decrease both in the amount of ND1 mRNA and co-transcribed tRNA^Leu(UUR) in mutant cells. Thus, mitochondrial dysfunctions caused by the T5655C and T3308C mutations may modulate the phenotypic manifestation of the T7511C mutation. These observations imply that a combination of the T7511C mutation with two mtDNA mutations accounts for the high penetrance of deafness in this family.     

Molecular basis for human mitochondrial tRNA m3C modification by alternatively spliced METTL8

METTL8 has recently been identified as the methyltransferase catalyzing 3-methylcytidine biogenesis at position 32 (m³C32) of mitochondrial tRNAs. METTL8 also potentially participates in mRNA methylation and R-loop biogenesis. How METTL8 plays multiple roles in distinct cell compartments and catalyzes mitochondrial tRNA m³C formation remain unclear. Here, we discovered that alternative mRNA splicing generated several isoforms of METTL8. One isoform (METTL8-Iso1) was targeted to mitochondria via an N-terminal pre-sequence, while another one (METTL8-Iso4) mainly localized to the nucleolus. METTL8-Iso1-mediated m³C32 modification of human mitochondrial tRNA^Thr (hmtRNA^Thr) was not reliant on t⁶A modification at A37 (t⁶A37), while that of hmtRNA^Ser(UCN) critically depended on i⁶A modification at A37 (i⁶A37). We clarified the hmtRNA^Thr substrate recognition mechanism, which was obviously different from that of hmtRNA^Ser(UCN), in terms of requiring a G35 determinant. Moreover, SARS2 (mitochondrial seryl-tRNA synthetase) interacted with METTL8-Iso1 in an RNA-independent manner and modestly accelerated m³C modification activity. We further elucidated how nonsubstrate tRNAs in human mitochondria were efficiently discriminated by METTL8-Iso1. In summary, our results established the expression pattern of METTL8, clarified the molecular basis for m³C32 modification by METTL8-Iso1 and provided the rationale for the involvement of METTL8 in tRNA modification, mRNA methylation or R-loop biogenesis.     

The complete mitochondrial genome of the leafminer <Emphasis Type="Italic">Liriomyza trifolii</Emphasis> (Diptera: Agromyzidae)

Liriomyza trifolii (Diptera: Agromyzidae) is one of the most economically significant pests in the world. In this paper we present sequence data for the complete mitochondrial genome of L. trifolii. The circular genome is 16,141 bp long and contains one encoding region including 37 genes and one non-coding A+T-rich region. Gene numbers and organization is similar to that of the typical insect mitochondrial genomes except that two additional tRNA genes are found in the A+T-rich region (tRNA^Thr and tRNA^Leu(UUR)). All of the protein initiation codons are ATN, except ND1 which begins with GTG and COI which is initiated by the quadruplet ATCA. The 22 tRNA anticodons of L. trifolii match those observed in Drosophila yakuba, and all of tRNAs form the typical cloverleaf structure except for tRNA^Ser(AGN), which has lost the DHU-arm. The A+T-rich region of L. trifolii also contains two previously noted Diperan features—a highly conserved polyT stretch and a (TA)_n stretch.     

NSUN3 and ABH1 modify the wobble position of mt‐tRNAMet to expand codon recognition in mitochondrial translation

Mitochondrial gene expression uses a non‐universal genetic code in mammals. Besides reading the conventional AUG codon, mitochondrial (mt‐)tRNA^Met mediates incorporation of methionine on AUA and AUU codons during translation initiation and on AUA codons during elongation. We show that the RNA methyltransferase NSUN3 localises to mitochondria and interacts with mt‐tRNA^Met to methylate cytosine 34 (C34) at the wobble position. NSUN3 specifically recognises the anticodon stem loop (ASL) of the tRNA, explaining why a mutation that compromises ASL basepairing leads to disease. We further identify ALKBH1/ABH1 as the dioxygenase responsible for oxidising m⁵C34 of mt‐tRNA^Met to generate an f⁵C34 modification. In vitro codon recognition studies with mitochondrial translation factors reveal preferential utilisation of m⁵C34 mt‐tRNA^Met in initiation. Depletion of either NSUN3 or ABH1 strongly affects mitochondrial translation in human cells, implying that modifications generated by both enzymes are necessary for mt‐tRNA^Met function. Together, our data reveal how modifications in mt‐tRNA^Met are generated by the sequential action of NSUN3 and ABH1, allowing the single mitochondrial tRNA^Met to recognise the different codons encoding methionine.     

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.     

An unusual tRNAThr derived from tRNAHis reassigns in yeast mitochondria the CUN codons to threonine

The standard genetic code is used by most living organisms, yet deviations have been observed in many genomes, suggesting that the genetic code has been evolving. In certain yeast mitochondria, CUN codons are reassigned from leucine to threonine, which requires an unusual tRNA^Thr with an enlarged 8-nt anticodon loop (). To trace its evolutionary origin we performed a comprehensive phylogenetic analysis which revealed that evolved from yeast mitochondrial tRNA^His. To understand this tRNA identity change, we performed mutational and biochemical experiments. We show that Saccharomyces cerevisiae mitochondrial threonyl-tRNA synthetase (MST1) could attach threonine to both and the regular , but not to the wild-type tRNA^His. A loss of the first nucleotide (G₋₁) in tRNA^His converts it to a substrate for MST1 with a K_m value (0.7 μM) comparable to that of (0.3 μM), and addition of G₋₁ to allows efficient histidylation by histidyl-tRNA synthetase. We also show that MST1 from Candida albicans, a yeast in which CUN codons remain assigned to leucine, could not threonylate , suggesting that MST1 has coevolved with . Our work provides the first clear example of a recent recoding event caused by alloacceptor tRNA gene recruitment.     

The Amyloid-β-SDR5C1(ABAD) Interaction Does Not Mediate a Specific Inhibition of Mitochondrial RNase P

The amyloid-β peptide (Aβ) is suggested to cause mitochondrial dysfunction in Alzheimer’s disease. The mitochondrial dehydrogenase SDR5C1 (also known as ABAD) was shown to bind Aβ and was proposed to thereby mediate mitochondrial toxicity, but the molecular mechanism has not been clarified. We recently identified SDR5C1 as an essential component of human mitochondrial RNase P and its associated tRNA:m¹R9 methyltransferase, the enzymes responsible for tRNA 5′-end processing and methylation of purines at tRNA position 9, respectively. With this work we investigated whether SDR5C1’s role as a subunit of these two tRNA-maturation activities represents the mechanistic link between Aβ and mitochondrial dysfunction. Using recombinant enzyme components, we tested RNase P and methyltransferase activity upon titration of Aβ. Micromolar concentrations of monomeric or oligomerized Aβ were required to inhibit tRNA 5′-end processing and position 9 methylation catalyzed by the SDR5C1-containing enzymes, yet similar concentrations of Aβ also inhibited related RNase P and methyltransferase activities, which do not contain an SDR5C1 homolog. In conclusion, the proposed deleterious effect of Aβ on mitochondrial function cannot be explained by a specific inhibition of mitochondrial RNase P or its tRNA:m¹R9 methyltransferase subcomplex, and the molecular mechanism of SDR5C1-mediated Aβ toxicity remains unclear.