Rapid Shifts in Mitochondrial tRNA Import in a Plant Lineage with Extensive Mitochondrial tRNA Gene Loss

In most eukaryotes, transfer RNAs (tRNAs) are one of the very few classes of genes remaining in the mitochondrial genome, but some mitochondria have lost these vestiges of their prokaryotic ancestry. Sequencing of mitogenomes from the flowering plant genus Silene previously revealed a large range in tRNA gene content, suggesting rapid and ongoing gene loss/replacement. Here, we use this system to test longstanding hypotheses about how mitochondrial tRNA genes are replaced by importing nuclear-encoded tRNAs. We traced the evolutionary history of these gene loss events by sequencing mitochondrial genomes from key outgroups (Agrostemma githago and Silene [=Lychnis] chalcedonica). We then performed the first global sequencing of purified plant mitochondrial tRNA populations to characterize the expression of mitochondrial-encoded tRNAs and the identity of imported nuclear-encoded tRNAs. We also confirmed the utility of high-throughput sequencing methods for the detection of tRNA import by sequencing mitochondrial tRNA populations in a species (Solanum tuberosum) with known tRNA trafficking patterns. Mitochondrial tRNA sequencing in Silene revealed substantial shifts in the abundance of some nuclear-encoded tRNAs in conjunction with their recent history of mt-tRNA gene loss and surprising cases where tRNAs with anticodons still encoded in the mitochondrial genome also appeared to be imported. These data suggest that nuclear-encoded counterparts are likely replacing mitochondrial tRNAs even in systems with recent mitochondrial tRNA gene loss, and the redundant import of a nuclear-encoded tRNA may provide a mechanism for functional replacement between translation systems separated by billions of years of evolutionary divergence.     

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.     

An evolutionary 'intermediate state' of mitochondrial translation systems found in Trichinella species of parasitic nematodes: co-evolution of tRNA and EF-Tu

EF-Tu delivers aminoacyl-tRNAs to ribosomes in the translation system. However, unusual truncations found in some animal mitochondrial tRNAs seem to prevent recognition by a canonical EF-Tu. We showed previously that the chromadorean nematode has two distinct EF-Tus, one of which (EF-Tu1) binds only to T-armless aminoacyl-tRNAs and the other (EF-Tu2) binds to D-armless Ser-tRNAs. Neither of the EF-Tus can bind to canonical cloverleaf tRNAs. In this study, by analyzing the translation system of enoplean nematode Trichinella species, we address how EF-Tus and tRNAs have evolved from the canonical structures toward those of the chromadorean translation system. Trichinella mitochondria possess three types of tRNAs: cloverleaf tRNAs, which do not exist in chromadorean nematode mitochondria; T-armless tRNAs; and D-armless tRNAs. We found two mitochondrial EF-Tu species, EF-Tu1 and EF-Tu2, in Trichinella britovi. T.britovi EF-Tu2 could bind to only D-armless Ser-tRNA, as Caenorhabditis elegans EF-Tu2 does. In contrast to the case of C.elegans EF-Tu1, however, T.britovi EF-Tu1 bound to all three types of tRNA present in Trichinella mitochondria. These results suggest that Trichinella mitochondrial translation system, and particularly the tRNA-binding specificity of EF-Tu1, could be an intermediate state between the canonical system and the chromadorean nematode mitochondrial system.     

Nucleotide sequences of animal mitochondrial tRNAs(Met) possibly recognizing both AUG and AUA codons.

To elucidate the role of modified nucleosides of tRNA in mitochondrial translation systems, especially with regard to their codon recognition, we purified mitochondrial tRNAs(Met) isolated from liver of frog, chicken and rat, and determined their nucleotide sequences. All of these tRNAs(Met) were found to possess 5-formylcytidine in the first letter of the anticodon, which is known to be prerequisite for bovine mt tRNA(Met) to decode AUA codon as well as AUG codon. These tRNA possesses two pseudeuridines in similar positions, and only chicken tRNA(Met) had ribothymidine at the first position of the T-loop, which is always found in the usual tRNAs. Considering that AUA codon is used as five times frequently as AUG codon in these animal mitochondrial genomes, it is deduced that 5-formylcytidine at the wobble position is essential for the recognition of both AUA and AUG codons.     

Mitochondrial translation is essential in bloodstream forms of Trypanosoma brucei

The parasitic protozoa Trypanosoma brucei has a complex life cycle. Oxidative phosphorylation is highly active in the procyclic form but absent from bloodstream cells. The mitochondrial genome encodes several gene products that are required for oxidative phosphorylation, but it completely lacks tRNA genes. For mitochondrial translation to occur, the import of cytosolic tRNAs is therefore essential for procyclic T. brucei. Whether the same is true for the bloodstream form has not been studied so far. Here we show that the steady-state levels of mitochondrial tRNAs are essentially the same in both life stages. Editing of the imported tRNA(Trp) also occurs in both forms as well as in mitochondria of Trypanosoma evansi, which lacks a genome and a translation system. These results show that mitochondrial tRNA import is a constitutive process that must be mediated by proteins that are expressed in both forms of the life cycle and that are not encoded in the mitochondrial genome. Moreover, bloodstream cells lacking either mitochondria-specific translation elongation factor Tu or mitochondrial tryptophanyl-tRNA synthetase are not viable indicating that mitochondrial translation is also essential in this stage. Both of these proteins show trypanosomatid-specific features and may therefore be excellent novel drug targets.     

Transfer RNA genes in the mitochondrial genome from a liverwort, Marchantia polymorpha: the absence of chloroplast-like tRNAs.

Twenty-nine genes for 27 species of tRNAs were deduced from the complete nucleotide sequence of the mitochondrial genome from a liverwort, Marchantia polymorpha. One to three species of tRNA genes corresponded to each of 20 amino acids including three species for leucine and arginine, two species for serine and glycine, and one for the rest of the amino acids. Interestingly, all tRNA genes were located in the semicircle of the liverwort mitochondrial genome except for the trnY and trnR genes. The region containing these tRNA genes was originally duplicated, and two trnR genes have diverged from each other. On the other hand, trnY and trnfM are present as two identical copies. The G:U and U:N wobbling between the first nucleotide of the anticodon and the third nucleotide of the codon permit the 27 tRNA identified species to translate almost all codons. However, at least two additional tRNA genes, trnl-GAU for AUY codon and trnT-UGU for ACR codon, are required to read all codons used in the liverwort mitochondrial genome. All of the identified tRNA genes are 'native' in liverwort mitochondria, not 'chloroplast-like' tRNAs as are found in the mitochondria of higher plants. This result implies that the tRNA gene transfer from chloroplast to mitochondrial genome in higher plants has occurred after the divergence from bryophytes.     

The human mitochondrial genome and evolution of transfer methionine RNA

The recently deciphered sequence of the human mitochondrial genome is analyzed in the light of an archigenetic hypothesis, according to which mitochondria are derived neither from pro- nor eukaryotes but from more primitive organisms. The possibility that animal mitochondria have only one gene both for elongator and initiator methionine tRNA is supported but C-A pair forming cytosine in the anticodon of these tRNAs is considered to be unmodified. The evolution of the gene and of the codon reading pattern of the methionine tRNA is discussed.     

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.     

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.     

Selection on the codon bias ofChlamydomonas reinhardtii chloroplast genes and the plantpsbA gene

Plant chloroplast genes have a codon use that reflects the genome compositional bias of a high A+T content with the single exception of the highly translatedpsbA gene which codes for the photosystem II D1 protein. The codon usage of plantpsbA corresponds more closely to the limited tRNA population of the chloroplast and is very similar to the codon use observed in the chloroplast genes of the green algaChlamydomonas reinhardtii. This pattern of codon use may be an adaptation for increased translation efficiency. A correspondence between codon use of plantpsbA andChlamydomonas chloroplast genes and the tRNAs coded by the chloroplast genome, however, is not observed in all synonymous codon groups. It is shown here that the degree of correspondence between codon use and tRNA population in different synonymous groups is correlated with the second codon position composition. Synonymous groups with an A or T at the second codon position have a high representation of codons for which a complementary tRNA is coded by the chloroplast genome. Those with a G or C at the second position have an increased representation of codons that bind a chloroplast tRNA by wobble. It is proposed that the difference between synonymous groups in terms of codon adaptation to the tRNA population in plantpsbA andChlamydomonas chloroplast genes may be the result of differences in second position composition.     

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).     

Adaptation of aminoacylation identity rules to mammalian mitochondria

Many mammalian mitochondrial aminoacyl-tRNA synthetases are of bacterial-type and share structural domains with homologous bacterial enzymes of the same specificity. Despite this high similarity, synthetases from bacteria are known for their inability to aminoacylate mitochondrial tRNAs, while mitochondrial enzymes do aminoacylate bacterial tRNAs. Here, the reasons for non-aminoacylation by a bacterial enzyme of a mitochondrial tRNA have been explored. A mutagenic analysis performed on in vitro transcribed human mitochondrial tRNA^Asp variants tested for their ability to become aspartylated by Escherichia coli aspartyl-tRNA synthetase, reveals that full conversion cannot be achieved on the basis of the currently established tRNA/synthetase recognition rules. Integration of the full set of aspartylation identity elements and stabilization of the structural tRNA scaffold by restoration of D- and T-loop interactions, enable only a partial gain in aspartylation efficiency. The sequence context and high structural instability of the mitochondrial tRNA are additional features hindering optimal adaptation of the tRNA to the bacterial enzyme. Our data support the hypothesis that non-aminoacylation of mitochondrial tRNAs by bacterial synthetases is linked to the large sequence and structural relaxation of the organelle encoded tRNAs, itself a consequence of the high rate of mitochondrial genome divergence.     

Chloroplast suppressors that act on a mitochondrial mutation in Chlamydomonas reinhardtii

We report genetic evidence supporting the existence of suppressor genes in the chloroplast that act on a mitochondrial mutation that impairs heterotrophic growth in the green alga Chlamydomonas reinhardtii. One of these suppressors also acts on a point mutation in the rbcL gene in the chloroplast. These results are consistent with previous data showing that mitochondrial protein synthesis depends on chloroplast protein synthesis in C. reinhardtii. The nature of the interaction between chloroplasts and mitochondria is discussed in light of the requirement for import of tRNAs by plant mitochondria.     

Structure and Evolution of the Atypical Mitochondrial Genome of <Emphasis Type="Italic">Armadillidium vulgare</Emphasis> (Isopoda,Crustacea)

The crustacean isopod Armadillidium vulgare is characterized by an unusual ∼42-kb-long mitochondrial genome consisting of two molecules co-occurring in mitochondria: a circular ∼28-kb dimer formed by two ∼14-kb monomers fused in opposite polarities and a linear ∼14-kb monomer. Here we determined the nucleotide sequence of the fundamental monomeric unit of A. vulgare mitochondrial genome, to gain new insight into its structure and evolution. Our results suggest that the junction zone between monomers of the dimer structure is located in or near the control region. Direct sequencing indicated that the nucleotide sequences of the different monomer units are virtually identical. This suggests that gene conversion and/or replication processes play an important role in shaping nucleotide sequence variation in this mitochondrial genome. The only heteroplasmic site we identified predicts an alloacceptor tRNA change from tRNA^Ala to tRNA^Val. Therefore, in A. vulgare, tRNA^Ala and tRNA^Val are found at the same locus in different monomers, ensuring that both tRNAs are present in mitochondria. The presence of this heteroplasmic site in all sequenced individuals suggests that the polymorphism is selectively maintained, probably because of the necessity of both tRNAs for maintaining proper mitochondrial functions. Thus, our results provide empirical evidence for the tRNA gene recruitment model of tRNA evolution. Moreover, interspecific comparisons showed that the A. vulgare mitochondrial gene order is highly derived compared to the putative ancestral arthropod type. By contrast, an overall high conservation of mitochondrial gene order is observed within crustacean isopods.     

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.     

Tissue-specific differences in human transfer RNA expression

Over 450 transfer RNA (tRNA) genes have been annotated in the human genome. Reliable quantitation of tRNA levels in human samples using microarray methods presents a technical challenge. We have developed a microarray method to quantify tRNAs based on a fluorescent dye-labeling technique. The first-generation tRNA microarray consists of 42 probes for nuclear encoded tRNAs and 21 probes for mitochondrial encoded tRNAs. These probes cover tRNAs for all 20 amino acids and 11 isoacceptor families. Using this array, we report that the amounts of tRNA within the total cellular RNA vary widely among eight different human tissues. The brain expresses higher overall levels of nuclear encoded tRNAs than every tissue examined but one and higher levels of mitochondrial encoded tRNAs than every tissue examined. We found tissue-specific differences in the expression of individual tRNA species, and tRNAs decoding amino acids with similar chemical properties exhibited coordinated expression in distinct tissue types. Relative tRNA abundance exhibits a statistically significant correlation to the codon usage of a collection of highly expressed, tissue-specific genes in a subset of tissues or tRNA isoacceptors. Our findings demonstrate the existence of tissue-specific expression of tRNA species that strongly implicates a role for tRNA heterogeneity in regulating translation and possibly additional processes in vertebrate organisms.     

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.