Differential import of nuclear-encoded tRNAGly isoacceptors into solanum Tuberosum mitochondria

In potato ( Solanum tuberosum ) mitochondria, about two-thirds of the tRNAs are encoded by the mitochondrial genome and one-third is imported from the cytosol. In the case of tRNAGly isoacceptors, a mitochondrial-encoded tRNAGly(GCC) was found in potato mitochondria, but this is likely to be insufficient to decode the four GGN glycine codons. In this work, we identified a cytosolic tRNAGly(UCC), which was found to be present in S.tuberosum mitochondria. The cytosolic tRNAGly(CCC) was also present in mitochondria, but to a lesser extent. By contrast, the cytosolic tRNAGly(GCC) could not be detected in mitochondria. This selective import of tRNAGly isoacceptors into S. tuberosum mitochondria raises further questions about the mechanism under-lying the specificity of the import process.     

The anticodon and the D-domain sequences are essential determinants for plant cytosolic tRNA(Val) import into mitochondria

In higher plants, one-third to one-half of the mitochondrial tRNAs are encoded in the nucleus and are imported into mitochondria. This process appears to be highly specific for some tRNAs, but the factors that interact with tRNAs before and/or during import, as well as the signals present on the tRNAs, still need to be identified. The rare experiments performed so far suggest that, besides the probable implication of aminoacyl-tRNA synthetases, at least one additional import factor and/or structural features shared by imported tRNAs must be involved in plant mitochondrial tRNA import. To look for determinants that direct tRNA import into higher plant mitochondria, we have transformed BY2 tobacco cells with Arabidopsis thaliana cytosolic tRNA(Val)(AAC) carrying various mutations. The nucleotide replacements introduced in this naturally imported tRNA correspond to the anticodon and/or D-domain of the non-imported cytosolic tRNA(Met-e). Unlike the wild-type tRNA(Val)(AAC), a mutant tRNA(Val) carrying a methionine CAU anticodon that switches the aminoacylation of this tRNA from valine to methionine is not present in the mitochondrial fraction. Furthermore, mutant tRNAs(Val) carrying the D-domain of the tRNA(Met-e), although still efficiently recognized by the valyl-tRNA synthetase, are not imported any more into mitochondria. These data demonstrate that in plants, besides identity elements required for the recognition by the cognate aminoacyl-tRNA synthetase, tRNA molecules contain other determinants that are essential for mitochondrial import selectivity. Indeed, this suggests that the tRNA import mechanism occurring in plant mitochondria may be different from what has been described so far in yeast or in protozoa.     

Overlapping destinations for two dual targeted glycyl-tRNA synthetases in Arabidopsis thaliana and Phaseolus vulgaris

In plant mitochondria, some of the tRNAs are encoded by the mitochondrial genome and resemble their prokaryotic counterparts, whereas the remaining tRNAs are encoded by the nuclear genome and imported from the cytosol. Generally, mitochondrial isoacceptor tRNAs all have the same genetic origin. One known exception to this rule is the group of tRNA(Gly) isoacceptors in dicotyledonous plants. A mitochondrion-encoded tRNA(Gly) and at least one nucleus-encoded tRNA(Gly) coexist in the mitochondria of these plants, and both are required to allow translation of all four GGN glycine codons. We have taken advantage of this atypical situation to address the problem of tRNA/aminoacyl-tRNA synthetase coevolution in plants. In this work, we show that two different nucleus-encoded glycyl-tRNA synthetases (GlyRSs) are imported into Arabidopsis thaliana and Phaseolus vulgaris mitochondria. The first one, GlyRS-1, is similar to human or yeast glycyl-tRNA synthetase, whereas the second, GlyRS-2, is similar to Escherichia coli glycyl-tRNA synthetase. Both enzymes are dual targeted, GlyRS-1 to mitochondria and to the cytosol and GlyRS-2 to mitochondria and chloroplasts. Unexpectedly, GlyRS-1 seems to be active in the cytosol but inactive in mitochondrial fractions, whereas GlyRS-2 is likely to glycylate both the organelle-encoded tRNA(Gly) and the imported tRNA(Gly) present in 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.     

Expression of Arabidopsis thaliana mitochondrial alanyl-tRNA synthetase is not sufficient to trigger mitochondrial import of tRNAAla in yeast

It has often been suggested that precursors to mitochondrial aminoacyl-tRNA synthetases are likely carriers for mitochondrial import of tRNAs in those organisms where this process occurs. In plants, it has been shown that mutation of U(70) to C(70) in Arabidopsis thaliana tRNA(Ala)(UGC) blocks aminoacylation and also prevents import of the tRNA into mitochondria. This suggests that interaction of tRNA(Ala) with alanyl-tRNA synthetase (AlaRS) is necessary for import to occur. To test whether this interaction is sufficient to drive import, we co-expressed A. thaliana tRNA(Ala)(UGC) and the precursor to the A. thaliana mitochondrial AlaRS in Saccharomyces cerevisiae. The A. thaliana enzyme and its cognate tRNA were correctly expressed in yeast in vivo. However, although the plant AlaRS was efficiently imported into mitochondria in the transformed strains, we found no evidence for import of the A. thaliana tRNA(Ala) nor of the endogenous cytosolic tRNA(Ala) isoacceptors. We conclude that at least one other factor besides the mitochondrial AlaRS precursor must be involved in mitochondrial import of tRNA(Ala) in plants.     

Mitochondrial import of only one of three nuclear-encoded glutamine tRNAs in Tetrahymena thermophila.

The mitochondrial genome of Tetrahymena does not appear to encode enough tRNAs to perform mitochondrial protein synthesis. It has therefore been proposed that nuclear-encoded tRNAs are imported into the mitochondria. T.thermophila has three major glutamine tRNAs: tRNA(Gln)(UUG), tRNA(Gln)(UUA) and tRNA(Gln)(CUA). Each of these tRNAs functions in cytosolic translation. However, due to differences between the Tetrahymena nuclear and mitochondrial genetic codes, only tRNA(Gln)(UUG) has the capacity to function in mitochondrial translation as well. Here we show that approximately 10-20% of the cellular complement of tRNA(Gln)(UUG) is present in mitochondrial RNA fractions, compared with 1% or less for the other two glutamine tRNAs. Furthermore, this glutamine tRNA is encoded only by a family of nuclear genes, the sequences of several of which are presented. Finally, when marked versions of tRNA(Gln)(UUG) and tRNA(Gln)(UUA) flanked by identical sequences are expressed in the macronucleus, only the former undergoes mitochondrial import; thus sequences within tRNA(Gln)(UUG) direct import. Because tRNA(Gln)(UUG) is a constituent of mitochondrial RNA fractions and is encoded only by nuclear genes, and because ectopically expressed tRNA(Gln)(UUG) fractionates with mitochondria like its endogenous counterpart, we conclude that it is an imported tRNA in T.thermophila.     

A single base change prevents import of cytosolic tRNAAla into mitochondria in transgenic plants

Plant mitochondria do not contain a full set of tRNA genes, and the additional tRNAs needed for protein synthesis (including tRNA^Ala) are imported from the cytosol. The import process appears to be highly specific for certain tRNAs, and it has been suggested that the cognate aminoacyl-tRNA synthetases may be responsible for this specificity. In order to test this, we have grown transgenic tobacco plants expressing Arabidopsis thaliana tRNA^Ala carrying a U₇₀ to C₇₀ mutation, which we have previously shown blocks aminoacylation by the plant alanyl-tRNA synthetase. Unlike the wild-type tRNA^Ala, the mutant tRNA is not present in the mitochondrial tRNA fraction. This is the first report of a tRNA mutation which prevents mitochondrial import and strongly supports the hypothesis that aminoacyl-tRNA synthetases are involved in this process in plants. Insertion of four bases into the anticodon loop of tRNA^Ala does not prevent mitochondrial import, implying that the tRNA might not need to participate in translation to be imported.     

The T-domain of cytosolic tRNAVal, an essential determinant for mitochondrial import

Import of tRNAs into plant mitochondria appears to be highly specific. We recently showed that the anticodon and the D-domain sequences are essential determinants for tRNAVal import into tobacco cell mitochondria. To determine the minimal set of elements required to direct import of a cytosol-specific tRNA species, tobacco cells were transformed with an Arabidopsis thaliana intron-containing tRNAMet-e gene carrying the D-domain and the anticodon of a valine tRNA. Although well expressed and processed into tobacco cells, this mutated tRNA was shown to remain in the cytosol. Furthermore, a mutant tRNAVal carrying the T-domain of the tRNAMet-e, although still efficiently recognized by the valyl-tRNA synthetase, is not imported into mitochondria. Altogether these results suggest that mutations affecting the core of a tRNA molecule also alter its import ability into plant mitochondria.     

Taurine-containing uridine modifications in tRNA anticodons are required to decipher non-universal genetic codes in ascidian mitochondria

Variations in the genetic code are found frequently in mitochondrial decoding systems. Four non-universal genetic codes are employed in ascidian mitochondria: AUA for Met, UGA for Trp, and AGA/AGG(AGR) for Gly. To clarify the decoding mechanism for the non-universal genetic codes, we isolated and analyzed mitochondrial tRNAs for Trp, Met, and Gly from an ascidian, Halocynthia roretzi. Mass spectrometric analysis identified 5-taurinomethyluridine (τm(5)U) at the anticodon wobble positions of tRNA(Met)(AUR), tRNA(Trp)(UGR), and tRNA(Gly)(AGR), suggesting that τm(5)U plays a critical role in the accurate deciphering of all four non-universal codes by preventing the misreading of pyrimidine-ending near-cognate codons (NNY) in their respective family boxes. Acquisition of the wobble modification appears to be a prerequisite for the genetic code alteration.     

Identification and structural characterization of nucleus-encoded transfer RNAs imported into wheat mitochondria

Despite its large size (200-2400 kilobase pairs), the mitochondrial genome of angiosperms does not encode the minimal set of tRNAs required to support mitochondrial protein synthesis. Here we report the identification of cytosolic-like tRNAs in wheat mitochondria using a method involving quantitative hybridization to distinguish among three tRNA classes: (i) those encoded by mitochondrial DNA (mtDNA) and localized in mitochondria, (ii) those encoded by nuclear DNA and located in the cytosol, and (iii) those encoded by nuclear DNA and found in both the cytosol and mitochondria. The latter class comprises tRNA species that are considered to be imported into mitochondria to compensate for the deficiency of mtDNA-encoded tRNAs. In a comprehensive survey of the wheat mitochondrial tRNA population, we identified 14 such imported tRNAs, the structural characterization of which is presented here. These imported tRNAs complement 16 mtDNA-encoded tRNAs, for a total of at least 30 distinct tRNA species in wheat mitochondria. Considering differences in the set of mtDNA-encoded and imported tRNAs in the mitochondria of various land plants, the import system must be able to adapt relatively rapidly over evolutionary time with regard to the particular cytosolic-like tRNAs that are brought into mitochondria.     

Induced tRNA Import into Human Mitochondria: Implication of a Host Aminoacyl-tRNA-Synthetase

In human cell, a subset of small non-coding RNAs is imported into mitochondria from the cytosol. Analysis of the tRNA import pathway allowing targeting of the yeast tRNA^Lys _CUU into human mitochondria demonstrates a similarity between the RNA import mechanisms in yeast and human cells. We show that the cytosolic precursor of human mitochondrial lysyl-tRNA synthetase (preKARS2) interacts with the yeast tRNA^Lys _CUU and small artificial RNAs which contain the structural elements determining the tRNA mitochondrial import, and facilitates their internalization by isolated human mitochondria. The tRNA import efficiency increased upon addition of the glycolytic enzyme enolase, previously found to be an actor of the yeast RNA import machinery. Finally, the role of preKARS2 in the RNA mitochondrial import has been directly demonstrated in vivo, in cultured human cells transfected with the yeast tRNA and artificial importable RNA molecules, in combination with preKARS2 overexpression or downregulation by RNA interference. These findings suggest that the requirement of protein factors for the RNA mitochondrial targeting might be a conserved feature of the RNA import pathway in different organisms.     

Recent advances in tRNA mitochondrial import

In many eukaryotes, tRNA import from the cytosol into mitochondria is essential for mitochondrial biogenesis and, consequently, for cell viability. Recent work has begun to unravel the molecular mechanisms involved in tRNA transport in yeast, trypanosomatids and plants. The mechanisms of tRNA targeting to, and translocation through, the double mitochondrial membrane in addition to how selectivity and regulation of these processes are achieved are the main questions that have been addressed. The characterization of both direct and co-import mechanisms involving distinct protein-import factors is in agreement with a polyphyletic origin of tRNA import. Moreover, our increased understanding of the tRNA-import pathway has been exploited recently to rescue dysfunctions associated with mitochondrial tRNA mutations.     

Transfer RNAs of potato (Solanum tuberosum) mitochondria have different genetic origins.

Total transfer RNAs were extracted from highly purified potato mitochondria. From quantitative measurements, the in vivo tRNA concentration in mitochondria was estimated to be in the range of 60 microM. Total potato mitochondrial tRNAs were fractionated by two-dimensional polyacrylamide gel electrophoresis. Thirty one individual tRNAs, which could read all sense codons, were identified by aminoacylation, sequencing or hybridization to specific oligonucleotides. The tRNA population that we have characterized comprises 15 typically mitochondrial, 5 'chloroplast-like' and 11 nuclear-encoded species. One tRNA(Ala), 2 tRNAs(Arg), 1 tRNA(Ile), 5 tRNAs(Leu) and 2 tRNAs(Thr) were shown to be coded for by nuclear DNA. A second, mitochondrial-encoded, tRNA(Ile) was also found. Five 'chloroplast-like' tRNAs, tRNA(Trp), tRNA(Asn), tRNA(His), tRNA(Ser)(GGA) and tRNA(Met)m, presumably transcribed from promiscuous chloroplast DNA sequences inserted in the mitochondrial genome, were identified, but, in contrast to wheat (1), potato mitochondria do not seem to contain 'chloroplast-like' tRNA(Cys) and tRNA(Phe). The two identified tRNAs(Val), as well as the tRNA(Gly), were found to be coded for by the mitochondrial genome, which again contrasts with the situation in wheat, where the mitochondrial genome apparently contains no tRNA(Val) or tRNA(Gly) gene (2).     

Correction of translational defects in patient-derived mutant mitochondria by complex-mediated import of a cytoplasmic tRNA

A variety of clinical disorders result from mutations in mitochondrial tRNA genes, leading to translational defects. We show here that a protein complex from the kinetoplastid protozoon Leishmania induces specific, ATP-dependent import of human cytoplasmic tRNA(1)(Lys) into human mitochondria in vitro. The imported tRNA undergoes efficient aminoacylation within the organelle and supports organellar protein synthesis. Moreover, translation in mitochondria from patients with myclonic epilepsy with ragged red fibers (MERRF) and Kearns-Sayre syndrome (KSS), containing mutant tRNA(Lys) genes, is stimulated to near-wild-type levels and the formation of aberrant polypeptides suppressed by complex-mediated import. These results suggest a novel way to introduce RNAs for the modulation of mitochondrial gene expression.     

Steady-state levels of imported tRNAs in Chlamydomonas mitochondria are correlated with both cytosolic and mitochondrial codon usages

The mitochondrial genome of Chlamydomonas reinhardtii only encodes three expressed tRNA genes, thus most mitochondrial tRNAs are likely imported. The sharing of tRNAs between chloroplasts and mitochondria has been speculated in this organism. We first demonstrate that no plastidial tRNA is present in mitochondria and that the mitochondrial translation mainly relies on the import of nucleus-encoded tRNA species. Then, using northern analysis, we show that the extent of mitochondrial localization for the 49 tRNA isoacceptor families encoded by the C. reinhardtii nuclear genome is highly variable. Until now the reasons for such variability were unknown. By comparing cytosolic and mitochondrial codon usage with the sub-cellular distribution of tRNAs, we provide unprecedented evidence that the steady-state level of a mitochondrial tRNA is linked not only to the frequency of the cognate codon in mitochondria but also to its frequency in the cytosol, then allowing optimal mitochondrial translation.     

tRNAs are imported into mitochondria of Trypanosoma brucei independently of their genomic context and genetic origin.

The mitochondrial genome of Trypanosoma brucei does not encode any identifiable tRNAs. Instead, mitochondrial tRNAs are synthesized in the nucleus and subsequently imported into mitochondria. In order to analyse the signals which target the tRNAs into the mitochondria, an in vivo import system has been developed: tRNA variants were expressed episomally and their import into mitochondria assessed by purification and nuclease treatment of the mitochondrial fraction. Three tRNA genes were tested in this system: (i) a mutated version of the trypanosomal tRNA(Tyr); (ii) a cytosolic tRNA(His) of yeast; and (iii) a human cytosolic tRNA(Lys). The tRNAs were expressed in their own genomic context, or containing various lengths of the 5'-flanking sequence of the trypanosomal tRNA(Tyr) gene. In all cases efficient import of each of the tRNAs was observed. We independently confirmed the mitochondrial import of the yeast tRNA(His), since in organello [alpha-32P]ATP-labelling of the 3'-end of the tRNA was inhibited by carboxyatractyloside, a highly specific inhibitor of the mitochondrial adenine nucleotide translocator. Import of heterologous tRNAs in their own genomic contexts supports the conclusion that no specific targeting signals are necessary to import tRNAs into mitochondria of T. brucei, but rather that the tRNA structure itself is sufficient to specify import.     

Evolution of macromolecular import pathways in mitochondria,hydrogenosomes and mitosomes

All eukaryotes require mitochondria for survival and growth. The origin of mitochondria can be traced down to a single endosymbiotic event between two probably prokaryotic organisms. Subsequent evolution has left mitochondria a collection of heterogeneous organelle variants. Most of these variants have retained their own genome and translation system. In hydrogenosomes and mitosomes, however, the entire genome was lost. All types of mitochondria import most of their proteome from the cytosol, irrespective of whether they have a genome or not. Moreover, in most eukaryotes, a variable number of tRNAs that are required for mitochondrial translation are also imported. Thus, import of macromolecules, both proteins and tRNA, is essential for mitochondrial biogenesis. Here, we review what is known about the evolutionary history of the two processes using a recently revised eukaryotic phylogeny as a framework. We discuss how the processes of protein import and tRNA import relate to each other in an evolutionary context.