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.     

In Vitro Import of a Nuclearly Encoded tRNA into the Mitochondrion of Trypanosoma brucei

All of the mitochondrial tRNAs of Trypanosoma brucei have been shown to be encoded in the nucleus and must be imported into the mitochondrion. The import of nuclearly encoded tRNAs into the mitochondrion has been demonstrated in a variety of organisms and is essential for proper function in the mitochondrion. An in vitro import assay has been developed to study the pathway of tRNA import in T. brucei. The in vitro system utilizes crude isolated trypanosome mitochondria and synthetic RNAs transcribed from a cloned nucleus-encoded tRNA gene cluster. The substrate, composed of tRNA(Ser) and tRNA(Leu), is transcribed in tandem with a 59-nucleotide intergenic region. The tandem tRNA substrate is imported rapidly, while the mature-size tRNA(Leu) fails to be imported in this system. These results suggest that the preferred substrate for tRNA import into trypanosome mitochondria is a precursor molecule composed of tandemly linked tRNAs. Import of the tandem tRNA substrate requires (i) a protein component that is associated with the surface of the mitochondrion, (ii) ATP pools both outside and within the mitochondrion, and (iii) a membrane potential. Dissipation of the proton gradient across the inner mitochondrial membrane by treatment with an uncoupling agent inhibits import of the tandem tRNA substrate. Characterization of the import requirements indicates that mitochondrial RNA import proceeds by a pathway including a protein component associated with the outer mitochondrial membrane, ATP-dependent steps, and a mitochondrial membrane potential.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Identification of nuclear encoded precursor tRNAs within the mitochondrion of Trypanosoma brucei.

RNAs that function in mitochondria are typically encoded by the mitochondrial DNA. However, the mitochondrial tRNAs of Trypanosoma brucei are encoded by the nuclear DNA and therefore must be imported into the mitochondrion. It is becoming evident that RNA import into mitochondria is phylogenetically widespread and is essential for cellular processes, but virtually nothing is known about the mechanism of RNA import. We have identified and characterized mitochondrial precursor tRNAs in T. brucei. The identification of mitochondrially located precursor tRNAs clearly indicates that mitochondrial tRNAs are imported as precursors. The mitochondrial precursor tRNAs hybridize to cloned nuclear tRNA genes, label with [alpha-32P]CTP using yeast tRNA nucleotidyltransferase and in isolated mitochondria via an endogenous nucleotidyltransferase-like activity, and are processed to mature tRNAs by Escherichia coli and yeast mitochondrial RNase P. We show that T. brucei mitochondrial extract contains an RNase P activity capable of processing a prokaryotic tRNA precursor as well as the T. brucei tRNA precursors. Precursors for tRNA(Asn) and tRNA(Leu) were detected on Northern blots of mitochondrial RNA, and the 5' ends of these RNAs were characterized by primer extension analysis. The structure of the precursor tRNAs and the significance of nuclear encoded precursor tRNAs within the mitochondrion are discussed.     

Striking differences in mitochondrial tRNA import between different plant species

A systematic comparison of the tRNAs imported into the mitochondria of larch, maize and potato reveals considerable differences among the three species. Larch mitochondria import at least eleven different tRNAs (more than half of those tested) corresponding to ten different amino acids. For five of these tRNAs [tRNA^Phe(GAA), tRNA^Lys(CUU), tRNA^Pro(UGG), tRNA^Ser(GCU) and tRNA^Ser(UGA)] this is the first report of import into mitochondria in any plant species. There are also differences in import between relatively closely related plants; wheat mitochondria, unlike maize mitochondria import tRNA^His, and sunflower mitochondria, unlike mitochondria from other angiosperms tested, import tRNA^Ser(GCU) and tRNA^Ser(UGA). These results suggest that the ability to import each tRNA has been acquired independently at different times during the evolution of higher plants, and that there are few apparent restrictions on which tRNAs can or cannot be imported. The implications for the mechanisms of mitochondrial tRNA Import in plants are discussed.     

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.     

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.     

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).     

tRNAs and Proteins Are Imported into Mitochondria of Trypanosoma brucei by Two Distinct Mechanisms

Import of tRNA into the mitochondrial matrix of Trypanosoma brucei was reconstituted in vitro. Efficient import required the hydrolysis of externally added ATP and was shown to be a carrier-mediated process depending on proteinaceous receptors on the surface of mitochondria. A partly synthetic tRNA(Tyr) as well as a physiological tRNA(Lys) were imported along the same pathway. Contrary to import of all matrix-localized proteins, tRNA import does not require a membrane potential. Furthermore, addition of an excess of import-competent tRNA had no effect on import of a mitochondrial matrix protein. In summary, these results show that tRNAs and proteins in T. brucei are imported by fundamentally different mechanisms.     

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.     

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.     

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.     

A sequence motif within trypanosome precursor tRNAs influences abundance and mitochondrial localization

Trypanosoma brucei lacks mitochondrial genes encoding tRNAs and must import nuclearly encoded tRNAs from the cytosol. The mechanism and specificity of this process remain unclear. We have identified a unique sequence motif, YGG(C/A)RRC, upstream of the genes encoding mitochondrially localized tRNAs in T. brucei. Both in vitro import studies and in vivo transfection studies indicate that deletion of the YGG(C/A)RRC sequence alters mitochondrial localization of tRNA(Leu), and in vivo studies also show a decrease in the cellular abundance of tRNA(Leu). These studies provide direct evidence for cis-acting RNA motifs within precursor tRNAs that facilitate the selection of tRNAs for mitochondrial import in trypanosomes. Furthermore, we found that mutations to the YGG(C/A)RRC sequence also altered the intracellular distribution of other endogenous tRNAs, suggesting a general role for this sequence in tRNA trafficking in trypanosomes.