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.     

Sequence dependence of tRNA(Gly) import into tobacco mitochondria

Plant mitochondrial genomes lack a number of tRNA genes and the corresponding tRNAs, which are nuclear-encoded, are imported from the cytosol. We show that specific import of tRNA(Gly) isoacceptors occurs in tobacco mitochondria: tRNA(Gly)(UCC) and tRNA(Gly)(CCC) are cytosolic and mitochondrial, while tRNA(Gly)(GCC) is found only in the cytosol. Exchange of sequences between tRNA(Gly)(UCC) and tRNA(Gly)(GCC) shows that the anticodon and D-domain are essential for tRNA(Gly)(UCC) import. However the reverse mutations in tRNA(Gly)(GCC) are not sufficient to promote its import into tobacco 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.     

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.     

End processing precedes mitochondrial importation and editing of tRNAs in Leishmania tarentolae

All mitochondrial tRNAs in Leishmania tarentolae are encoded in the nuclear genome and imported into the mitochondrion from the cytosol. One imported tRNA (tRNA(Trp)) is edited by a C to U modification at the first position of the anticodon. To determine the in vivo substrates for mitochondrial tRNA importation as well as tRNA editing, we examined the subcellular localization and extent of 5'- and 3'-end maturation of tRNA(Trp)(CCA), tRNA(Ile)(UAU), tRNA(Gln)(CUG), tRNA(Lys)(UUU), and tRNA(Val)(CAC). Nuclear, cytosolic, and mitochondrial fractions were obtained with little cross-contamination, as determined by Northern analysis of specific marker RNAs. tRNA(Gln) was mainly cytosolic in localization; tRNA(Ile) and tRNA(Lys) were mainly mitochondrial; and tRNA(Trp) and tRNA(Val) were shared between the two compartments. 5'- and 3'-extended precursors of all five tRNAs were present only in the nuclear fraction, suggesting that the mature tRNAs represent the in vivo substrates for importation into the mitochondrion. Consistent with this model, T7-transcribed mature tRNA(Ile) underwent importation in vitro into isolated mitochondria more efficiently than 5'-extended precursor tRNA(Ile). 5'-Extended precursor tRNA(Trp) was found to be unedited, which is consistent with a mitochondrial localization of this editing reaction. T7-transcribed unedited tRNA(Trp) was imported in vitro more efficiently than edited tRNA(Trp), suggesting the presence of importation determinants in the anticodon.     

Nuclear-encoded mitochondrial tRNAs of Trypanosoma brucei have a modified cytidine in the anticodon loop.

The mitochondrial genome of Trypanosoma brucei does not appear to encode any tRNA genes. Isolated organellar tRNAs hybridize to nuclear DNA, suggesting that they are synthesized in the nucleus and subsequently imported into the mitochondrion. Most imported tRNAs have cytosolic counterparts, showing identical mobility on two-dimensional polyacrylamide gels. We have compared three nuclear-encoded mitochondrial tRNAs (tRNA(Lys), tRNA(Leu), tRNA(Tyr)) with their cytosolic isoforms by direct enzymatic sequence analysis. Our findings indicate that the primary sequences of the mitochondrial and the corresponding cytosolic tRNAs are identical. However, we have identified a mitochondrion-specific nucleotide modification of each tRNA which is localized to a conserved cytidine residue at the penultimate position 5' of the anticodon. The modification present in mature mitochondrial tRNA(Tyr) was not found in a mutant tRNA(Tyr) defective in splicing in either cytosolic or mitochondrial fractions. The mutant tRNA(Tyr) has been expressed in transformed cells and its import into mitochondria has been demonstrated, suggesting that the modified cytidine residue is not required for import and therefore may be involved in adapting imported tRNAs to specific requirements of the mitochondrial translation machinery.     

Targeting of tRNA into yeast and human mitochondria: the role of anticodon nucleotides

In vivo, yeast mitochondria import a single cytoplasmic tRNA, tRNA(CUU)Lys, while human mitochondria do not import any cytoplasmic tRNA. We have previously demonstrated that both yeast and human isolated mitochondria can specifically internalize tRNA(CUU)Lys, several of its mutant versions and some mutant versions of yeast cytosolic tRNA(UUU)Lys (not imported in vivo). Aminoacylation of tRNA(CUU)Lys by the cytoplasmic lysyl-tRNA synthetase was a prerequisite for its import. Here we are studying the influence of one-base replacements in the anticodon of tRNAs(Lys) on their aminoacylation, on binding to the precursor of the mitochondrial lysyl-tRNA synthetase (carrier protein directing the import), and on the efficiency of import into isolated yeast and human mitochondria. We show that the base U35 is the main identity element for the yeast cytoplasmic lysyl-tRNA synthetase. The single replacement that abolished import was C34G, while all the others only modulated the import efficiency. The need of aminoacylation for import and for interaction with the carrier protein was shown only for a subset of mutant versions, while the others could be recognized and internalized without aminoacylation or in misacylated forms.     

Cytosolic yeast tRNA(His) is covalently modified when imported into mitochondria of Trypanosoma brucei.

The mitochondrial genome of Trypanosoma brucei does not encode any tRNAs. Instead, mitochondrial tRNAs are synthesized in the nucleus and subsequently imported into mitochondria. The great majority of mitochondrial tRNAs have cytosolic counterparts showing identical primary sequences. The only difference found between mitochondrial and cytosolic isotypes of the tRNAs are mitochondria-specific nucleotide modifications which appear to be a common feature of imported tRNAs in trypanosomes. In this study, a mutated yeast cytosolic tRNAHis was expressed in trypanosomes and its import phenotype was analyzed by cell fractionation and nuclease treatment of intact mitochondria. Furthermore, cytosolic and mitochondrial isotypes of the yeast tRNA(His) were specifically labeled and analyzed by limited alkaline hydrolysis. These experiments revealed the presence of mitochondria-specific nucleotide modifications in the yeast tRNA(His). The positions of the modifications were determined by direct enzymatic sequencing of the tRNA(His) and shown to correspond to the ultimate and penultimate nucleotides before the anticodon, the same relative positions which are modified in the mitochondrial isotype of trypanosomal tRNA(Tyr). The results demonstrate that covalent modification of tRNAs; in trypanosomal mitochondria can be used, in analogy to processing of precursor proteins during mitochondrial protein import, as a marker for import of both endogenous and heterologous tRNAs.     

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.     

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.     

Coexistence of nuclear DNA-encoded tRNAVal(AAC) and mitochondrial DNA-encoded tRNAVal(UAC) in mitochondria of a liverwort Marchantia polymorpha.

The liverwort Marchantia polymorpha mitochondrial DNA encodes almost all tRNAs required for mitochondrial translation except for the isoleucine (AUU, AUC) and threonine (ACA, ACG) codons, while the missing tRNAs are supplied in part by the nucleus and imported in mitochondria. In this paper, we report a finding of two radically different nuclear tRNAVal(AAC) genes and import of the corresponding tRNA isoacceptors in M.polymorpha mitochondria. This finding is surprising since the mtDNA encodes the gene for tRNAVal(UAC), which alone was considered sufficient for translating all four valine codons GUN by the U/N wobble mechanism. The present results suggest for the first time that the import of ncDNA-encoded tRNAs may result in decoding overlaps in plant mitochondria. The coexistence of nuclear DNA-encoded tRNAVal(AAC) and mitochondrial DNA-encoded tRNAVal(UAC) in liverwort mitochondria and the significance for the decoding mechanism as well as evolution of tRNA import are discussed.     

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.     

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.     

Does the evolutionary history of aminoacyl-tRNA synthetases explain the loss of mitochondrial tRNA genes?

The importation of cytosolic tRNAs is required for protein synthesis in the mitochondria of the wide variety of eukaryotes that lack a complete set of mitochondrial tRNA genes. The evolutionary history of the process, however, is still enigmatic. The analysis presented here suggests that the loss of distinct mitochondrial tRNA genes was not random and that it might be explained by the differential capabilities of mitochondrial aminoacyl-tRNA synthetases to charge imported eukaryotic-type tRNAs with amino acid.     

Mitochondrial RNA import in Leishmania tropica: aptamers homologous to multiple tRNA domains that interact cooperatively or antagonistically at the inner membrane

A large number of cytoplasmic tRNAs are imported into the kinetoplast-mitochondrion of Leishmania by a receptor-mediated process. To identify the sequences recognized by import receptors, mitochondria were incubated with a combinatorial RNA library. Repeated cycles of amplification of the imported sequences (SELEX) resulted in rapid selection of several import aptamers containing sequence motifs present in the anticodon arm, the D arm, the V-T region, and acceptor stem of known tRNAs, confirming or suggesting the presence of import signals in these domains. As predicted, truncated derivatives of tRNA(Ile)(UAU) containing the D arm or the V-T region were imported in vitro. Four aptamers were studied in detail. All were imported in vitro as well as in transiently transfected cells, using the same pathway as tRNA, but their individual import efficiencies were different. Two types of aptamers were discernible: the A arm and D arm homologues (type I), which were efficiently transferred across the inner mitochondrial membrane, and the V-T homologues (type II), which were not. Remarkably, subnanomolar concentrations of type I RNAs stimulated the entry of type II RNAs into the matrix, whereas type II RNAs inhibited inner membrane transfer of type I RNAs. Moreover, tRNA(Tyr)(GUA) and tRNA(Ile)(UAU) interacted with one another as type I and type II, respectively. Such cooperative and antagonistic interactions may allow the use of a limited number of receptors to recognize a large number of tRNAs of variable affinity and enable the maintenance of a properly balanced tRNA pool for mitochondrial translation.     

Thiolated tRNAs of Trypanosoma brucei Are Imported into Mitochondria and Dethiolated after Import

All mitochondrial tRNAs in Trypanosoma brucei derive from cytosolic tRNAs that are in part imported into mitochondria. Some trypanosomal tRNAs are thiolated in a compartment-specific manner. We have identified three proteins required for the thio modification of cytosolic tRNA^Gln, tRNA^Glu, and tRNA^Lys. RNA interference-mediated ablation of these proteins results in the cytosolic accumulation non-thio-modified tRNAs but does not increase their import. Moreover, in vitro import experiments showed that both thio-modified and non-thio-modified tRNA^Glu can efficiently be imported into mitochondria. These results indicate that unlike previously suggested the cytosol-specific thio modifications do not function as antideterminants for mitochondrial tRNA import. Consistent with these results we showed by using inducible expression of a tagged tRNA^Glu that it is mainly the thiolated form that is imported in vivo. Unexpectedly, the imported tRNA becomes dethiolated after import, which explains why the non-thiolated form is enriched in mitochondria. Finally, we have identified two genes required for thiolation of imported tRNA^Trp whose wobble nucleotide is subject to mitochondrial C to U editing. Interestingly, down-regulation of thiolation resulted in an increase of edited tRNA^Trp but did not affect growth.     

Evidence for an adaptation mechanism of mitochondrial translation via tRNA import from the cytosol

Although mitochondrial import of nuclear DNA-encoded RNAs is widely occurring, their functions in the organelles are not always understood. Mitochondrial function(s) of tRNA(Lys)(CUU), tRK1, targeted into Saccharomyces cerevisiae mitochondria was mysterious, since mitochondrial DNA-encoded tRNA(Lys)(UUU), tRK3, was hypothesized to decode both lysine codons, AAA and AAG. Mitochondrial targeting of tRK1 depends on the precursor of mitochondrial lysyl-tRNA synthetase, pre-Msk1p. Here we show that substitution of pre-Msk1p by its Ashbya gossypii ortholog results in a strain in which tRK3 is aminoacylated, while tRK1 is not imported. At elevated temperature, drop of tRK1 import inhibits mitochondrial translation of mRNAs containing AAG codons, which coincides with the impaired 2-thiolation of tRK3 anticodon wobble nucleotide. Restoration of tRK1 import cures the translational defect, suggesting the role of tRK1 in conditional adaptation of mitochondrial protein synthesis. In contrast with the known ways of organellar translation control, this mechanism exploits the RNA import pathway.