The nucleotide sequences of several tRNA genes from rat mitochondria: common features and relatedness to homologous species.

We have determined the nucleotide sequences of thirteen rat mt tRNA genes. The features of the primary and secondary structures of these tRNAs show that those for Gln, Ser, and f-Met resemble, while those for Lys, Cys, and Trp depart strikingly from the universal type. The remainder are slightly abnormal. Among many mammalian mt DNA sequences, those of mt tRNA genes are highly conserved, thus suggesting for those genes an additional, perhaps regulatory, function. A simple evolutionary relationship between the tRNAs of animal mitochondria and those of eukaryotic cytoplasm, of lower eukaryotic mitochondria or of prokaryotes, is not evident owing to the extreme divergence of the tRNA sequences in the two groups. However, a slightly higher homology does exist between a few animal mt tRNAs and those from prokaryotes or from lower eukaryotic mitochondria.     

The complete mitochondrial genome of yarrowia lipolytica

We here report the complete nucleotide sequence of the 47.9 kb mitochondrial (mt) genome from the obligate aerobic yeast Yarrowia lipolytica. It encodes, all on the same strand, seven subunits of NADH: ubiquinone oxidoreductase (ND1-6, ND4L), apocytochrome b (COB), three subunits of cytochrome oxidase (COX1, 2, 3), three subunits of ATP synthetase (ATP6, 8 and 9), small and large ribosomal RNAs and an incomplete set of tRNAs. The Y. lipolytica mt genome is very similar to the Hansenula wingei mt genome, as judged from blocks of conserved gene order and from sequence homology. The extra DNA in the Y. lipolytica mt genome consists of 17 group 1 introns and stretches of A+Trich sequence, interspersed with potentially transposable GC clusters. The usual mould mt genetic code is used. Interestingly, there is no tRNA able to read CGN (arginine) codons. CGN codons could not be found in exonic open reading frames, whereas they do occur in intronic open reading frames. However, several of the intronic open reading frames have accumulated mutations and must be regarded as pseudogenes. We propose that this may have been triggered by the presence of untranslatable CGN codons. This sequence is available under EMBL Accession No. AJ307410.     

The yeast model suggests the use of short peptides derived from mt LeuRS for the therapy of diseases due to mutations in several mt tRNAs

We have previously established a yeast model of mitochondrial (mt) diseases. We showed that defective respiratory phenotypes due to point-mutations in mt tRNA^Leu(UUR), tRNA^Ile and tRNA^Val could be relieved by overexpression of both cognate and non-cognate nuclearly encoded mt aminoacyl-tRNA synthetases (aaRS) LeuRS, IleRS and ValRS. More recently, we showed that the isolated carboxy-terminal domain (Cterm) of yeast mt LeuRS, and even short peptides derived from the human Cterm, have the same suppressing abilities as the whole enzymes.In this work, we extend these results by investigating the activity of a number of mt aaRS from either class I or II towards a panel of mt tRNAs. The Cterm of both human and yeast mt LeuRS has the same spectrum of activity as mt aaRS belonging to class I and subclass a, which is the most extensive among the whole enzymes. Yeast Cterm is demonstrated to be endowed with mt targeting activity.Importantly, peptide fragments β30_31 and β32_33, derived from the human Cterm, have even higher efficiency as well as wider spectrum of activity, thus opening new avenues for therapeutic intervention. Bind-shifting experiments show that the β30_31 peptide directly interacts with human mt tRNA^Leu(UUR) and tRNA^Ile, suggesting that the rescuing activity of isolated peptide fragments is mediated by a chaperone-like mechanism.Wide-range suppression appears to be idiosyncratic of LeuRS and its fragments, since it is not shared by Cterminal regions derived from human mt IleRS or ValRS, which are expected to have very different structures and interactions with tRNAs.     

A role for the bulged nucleotide 47 in the facilitation of tertiary interactions in the tRNA structure.

Based on computer modeling and with the use of energy minimisation procedure, we show that the bulged nucleotide 47 in the yeast tRNA(Phe) structure plays an important steric role, allowing the formation of canonical tertiary interactions 15-48 and 22-46 within the D-domain. The absence of nucleotide 47 can be compensated by the presence of a wobble pair U13-G22, whose unusual stereochemistry permits as well the formation of the canonical tertiary interactions. The tRNA database show that the vast majority of the cytosolic tRNAs have either a nucleotide at position 47 or a wobble pair U13-G22. On the contrary, many mitochondrial tRNAs, having a Watson-Crick pair 13-22, do not have nucleotide in position 47, which suggests that their tertiary interactions within the D-domain must differ from those in cytosolic tRNAs.     

Polymorphisms in the mitochondrial ribosome recycling factor EF-G2mt/MEF2 compromise cell respiratory function and increase atorvastatin toxicity

Mitochondrial translation, essential for synthesis of the electron transport chain complexes in the mitochondria, is governed by nuclear encoded genes. Polymorphisms within these genes are increasingly being implicated in disease and may also trigger adverse drug reactions. Statins, a class of HMG-CoA reductase inhibitors used to treat hypercholesterolemia, are among the most widely prescribed drugs in the world. However, a significant proportion of users suffer side effects of varying severity that commonly affect skeletal muscle. The mitochondria are one of the molecular targets of statins, and these drugs have been known to uncover otherwise silent mitochondrial mutations. Based on yeast genetic studies, we identify the mitochondrial translation factor MEF2 as a mediator of atorvastatin toxicity. The human ortholog of MEF2 is the Elongation Factor Gene (EF-G) 2, which has previously been shown to play a specific role in mitochondrial ribosome recycling. Using small interfering RNA (siRNA) silencing of expression in human cell lines, we demonstrate that the EF-G2mt gene is required for cell growth on galactose medium, signifying an essential role for this gene in aerobic respiration. Furthermore, EF-G2mt silenced cell lines have increased susceptibility to cell death in the presence of atorvastatin. Using yeast as a model, conserved amino acid variants, which arise from non-synonymous single nucleotide polymorphisms (SNPs) in the EF-G2mt gene, were generated in the yeast MEF2 gene. Although these mutations do not produce an obvious growth phenotype, three mutations reveal an atorvastatin-sensitive phenotype and further analysis uncovers a decreased respiratory capacity. These findings constitute the first reported phenotype associated with SNPs in the EF-G2mt gene and implicate the human EF-G2mt gene as a pharmacogenetic candidate gene for statin toxicity in humans.     

Primary structure of three tRNAs from brewer's yeast: tRNAPro2, tRNAHis1 and tRNAHis2

The primary structures of three brewer's yeast tRNAs: tRNAPro2 and tRNAHis1 and 2 have been determined (Formula:see text) The U* in the anticodon U*-G-G of tRNAPro2 is probably a derivative of U; tRNAPro2 has 80 per cent homology with mammalian tRNAsPro. tRNAHis1 and tRNAHis2 differ by only 5 nucleotides; they have identical anticodons and may therefore recognize both codons for histidine; they have an additional nucleotide at the 5' end. As in all other sequenced tRNAsHis this nucleotide is not paired with the fourth nucleotide from acceptor adenosine. All three sequenced tRNAs have a low degree of homology with their counterparts from yeast mitochondria.     

Wobble modification differences and subcellular localization of tRNAs in Leishmania tarentolae: implication for tRNA sorting mechanism

In Leishmania tarentolae, all mitochondrial tRNAs are encoded in the nuclear genome and imported from the cytosol. It is known that tRNA(Glu)(UUC) and tRNA(Gln)(UUG) are localized in both cytosol and mitochondria. We investigated structural differences between affinity-isolated cytosolic (cy) and mitochondrial (mt) tRNAs for glutamate and glutamine by mass spectrometry. A unique modification difference in both tRNAs was identified at the anticodon wobble position: cy tRNAs have 5-methoxycarbonylmethyl-2- thiouridine (mcm(5)s(2)U), whereas mt tRNAs have 5- methoxycarbonylmethyl-2'-O-methyluridine (mcm(5)Um). In addition, a trace portion (4%) of cy tRNAs was found to have 5-methoxycarbonylmethyluridine (mcm(5)U) at its wobble position, which could represent a common modification intermediate for both modified uridines in cy and mt tRNAs. We also isolated a trace amount of mitochondria-specific tRNA(Lys)(UUU) from the cytosol and found mcm(5)U at its wobble position, while its mitochondrial counterpart has mcm(5)Um. Mt tRNA(Lys) and in vitro transcribed tRNA(Glu) were imported much more efficiently into isolated mitochondria than the native cy tRNA(Glu) in an in vitro importation experiment, indicating that cytosol-specific 2-thiolation could play an inhibitory role in tRNA import into mitochondria.     

Yeast mitochondrial methionine initiator tRNA: characterization and nucleotide sequence.

Two methionine tRNAs from yeast mitochondria have been purified. The mitochondrial initiator tRNA has been identified by formylation using a mitochondrial enzyme extract. E. coli transformylase however, does not formylate the yeast mitochondrial initiator tRNA. The sequence was determined using both 32P-in vivo labeled and 32P-end labeled mt tRNAf(Met). This tRNA, unlike N. crassa mitochondrial tRNAf(Met), has two structural features typical of procaryotic initiator tRNAs: (i) it lacks a Watson-Crick base-pair at the end of the acceptor stem and (ii) has a T-psi-C-A sequence in loop IV. However, both yeast and N. crassa mitochondrial initiator tRNAs have a U11:A24 base-pair in the D-stem unlike procaryotic initiator tRNAs which have A11:U24. Interestingly, both mitochondrial initiator tRNAs, as well as bean chloroplast tRNAf(Met), have only two G:C pairs next to the anticodon loop, unlike any other initiator tRNA whatever its origin. In terms of overall sequence homology, yeast mitochondrial tRNA(Met)f differs from both procaryotic or eucaryotic initiator tRNAs, showing the highest homology with N. crassa mitochondrial initiator tRNA.     

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.     

The complete nucleotide sequence and gene organization of the mitochondrial genome of the bumblebee, Bombus ignitus (Hymenoptera: Apidae)

The complete 16,434-bp nucleotide sequence of the mitogenome of the bumble bee, Bombus ignitus (Hymenoptera: Apidae), was determined. The genome contains the base composition and codon usage typical of metazoan mitogenomes. An unusual feature of the B. ignitus mitogenome is the presence of five tRNA-like structures: two each of the tRNALeu(UUR)-like and tRNASer(AGN)-like sequences and one tRNAPhe-like sequence. These tRNA-like sequences have proper folding structures and anticodon sequences, but their functionality in their respective amino acid transfers remained uncertain. Among these sequences, the tRNALeu(UUR)-like sequence and the tRNASer(AGN)-like sequence are seemingly located within the A+T-rich region. This tRNASer(AGN)-like sequence is highly unusual in that its sequence homology is very high compared to the tRNAMet of other insects, including Apis mellifera, but it contains the anticodon ACT, which designates it as tRNASer(AGN). All PCG and rRNAs are conserved in positions observed most frequently in insect mitogenome structures, but the positions of the tRNAs are highly variable, presenting a new arrangement for an insect mitogenome. As a whole, the B. ignitus mitogenome contains the highest A+T content (86.9%) found in any of the complete insects mt sequences determined to date. All protein-coding sequences started with a typical ATN codon. Nine of the 13 PCGs have a complete termination codon (all TAA), but the remaining four genes terminate with the incomplete TA or T. All tRNAs have the typical clover-leaf structures of mt tRNAs, except for tRNASer(AGN), in which the DHU arm forms a simple loop. All anticodons of B. ignitus tRNAs are identical to those of A. mellifera. In the A+T-rich region, a highly conserved sequence block that was previously described in Orthoptera and Diptera was also present. The stem-and-loop structures that may play a role in the initiation of mtDNA replication were also found in this region. Phylogenetic analysis among three corbiculate tribes, represented by Melipona bicolor (Meliponini), A. mellifera (Apini), and B. ignitus (Bombini), showed the closest relationship between M. bicolor and B. ignitus.     

Identification and characterization of mammalian mitochondrial tRNA nucleotidyltransferases

The CCA-adding enzyme (ATP:tRNA adenylyltransferase or CTP:tRNA cytidylyltransferase (EC )) generates the conserved CCA sequence responsible for the attachment of amino acid at the 3' terminus of tRNA molecules. It was shown that enzymes from various organisms strictly recognize the elbow region of tRNA formed by the conserved D- and T-loops. However, most of the mammalian mitochondrial (mt) tRNAs lack consensus sequences in both D- and T-loops. To characterize the mammalian mt CCA-adding enzymes, we have partially purified the enzyme from bovine liver mitochondria and determined cDNA sequences from human and mouse dbESTs by mass spectrometric analysis. The identified sequences contained typical amino-terminal peptides for mitochondrial protein import and had characteristics of the class II nucleotidyltransferase superfamily that includes eukaryotic and eubacterial CCA-adding enzymes. The human recombinant enzyme was overexpressed in Escherichia coli, and its CCA-adding activity was characterized using several mt tRNAs as substrates. The results clearly show that the human mt CCA-adding enzyme can efficiently repair mt tRNAs that are poor substrates for the E. coli enzyme although both enzymes work equally well on cytoplasmic tRNAs. This suggests that the mammalian mt enzymes have evolved so as to recognize mt tRNAs with unusual structures.     

Gene recruitment--a common mechanism in the evolution of transfer RNA gene families

The evolution of alloacceptor transfer RNAs (tRNAs) has been traditionally thought to occur vertically and reflect the evolution of the genetic code. Yet there have been several indications that a tRNA gene could evolve horizontally, from a copy of an alloacceptor tRNA gene in the same genome. Earlier, we provided the first unambiguous evidence for the occurrence of such "tRNA gene recruitment" in nature--in the mitochondrial (mt) genome of the demosponge Axinella corrugata. Yet the extent and the pattern of this process in the evolution of tRNA gene families remained unclear. Here we analyzed tRNA genes from 21 mt genomes of demosponges as well as nuclear genomes of rhesus macaque, chimpanzee and human. We found four new cases of alloacceptor tRNA gene recruitment in mt genomes and eleven cases in the nuclear genomes. In most of these cases we observed a single nucleotide substitution at the middle position of the anticodon, which resulted in the change of not only the tRNA's amino-acid identity but also the class of the amino-acyl tRNA synthetases (aaRSs) involved in amino-acylation. We hypothesize that the switch to a different class of aaRSs may have prevented the conflict between anticodon and amino-acid identities of recruited tRNAs. Overall our results suggest that gene recruitment is a common phenomenon in tRNA multigene family evolution and should be taken into consideration when tRNA evolutionary history is reconstructed.     

Insertional editing in mitochondria of Physarum

RNA produced from a number of genes on the mitochondrial (mt) DNA of Physarum polycephalum have nucleotides inserted at specific sites in their sequence. These insertions are spaced at approximately 25 nucleotide intervals and create open reading frames in mRNA and functional structure in tRNAs and rRNAs. Although most of the insertions at a site are single cytidines; single uridines and certain dinucleotides containing adenosine and guanosine as well as cytidine and uridine are also occasionally inserted at certain sites. This mixed nucleotide insertional RNA editing is unique among currently characterized editing systems.     

Nucleotide sequences of the mitochondrial genes trnS(TGA) encoding tRNA(TGASer) in Oenothera berteriana and Arabidopsis thaliana

The genes encoding tRNA(TGASer) have been investigated in the mitochondrial (mt) genomes of Oenothera berteriana and Arabidopsis thaliana. Sequence analysis shows four nucleotide (nt) differences between the two dicots, but only two differences between each dicot and the available monocot sequences. Similarity comparisons identify these genes as encoding a native mt tRNA(TGASer), with less than 77% of the nt identical to the corresponding chloroplast tRNAs.