Adenosine-to-inosine editing in the anticodon of tRNAs is essential for viability. Enzymes mediating tRNA adenosine deamination in bacteria and yeast contain cytidine deaminase-conserved motifs, suggesting an evolutionary link between the two reactions. In trypanosomatids, tRNAs undergo both cytidine-to-uridine and adenosine-to-inosine editing, but the relationship between the two reactions is unclear. Here we show that down-regulation of the Trypanosoma brucei tRNA-editing enzyme by RNAi leads to a reduction in both C-to-U and A-to-I editing of tRNA in vivo. Surprisingly, in vitro, this enzyme can mediate A-to-I editing of tRNA and C-to-U deamination of ssDNA but not both in either substrate. The ability to use both DNA and RNA provides a model for a multispecificity editing enzyme. Notably, the ability of a single enzyme to perform two different deamination reactions also suggests that this enzyme still maintains specificities that would have been found in the ancestor deaminase, providing a first line of evidence for the evolution of editing deaminases.deaminases ͉ evolution ͉ hypermutation ͉ modification ͉ decoding

Mitochondrial genomes generally encode a minimal set of tRNAs necessary for protein synthesis. However, a number of eukaryotes import tRNAs from the cytoplasm into their mitochondria. For instance, Saccharomyces cerevisiae imports cytoplasmic tRNA Gln into the mitochondrion without any added protein factors. Here, we examine the existence of a similar active tRNA import system in mammalian mitochondria. We have used subcellular RNA fractions from rat liver and human cells to perform RT-PCR with oligonucleotide primers specific for nucleus-encoded tRNA CUG Gln and tRNA UUG Gln species, and we show that these tRNAs are present in rat and human mitochondria in vivo. Import of in vitro transcribed tRNAs, but not of heterologous RNAs, into isolated mitochondria also demonstrates that this process is tRNA-specific and does not require the addition of cytosolic factors. Although this in vitro system requires ATP, it is resistant to inhibitors of the mitochondrial electrochemical gradient, a key component of protein import. tRNA Gln import into mammalian mitochondria proceeds by a mechanism distinct from protein import. We also show that both tRNA Gln species and a bacterial pre-tRNA Asp can be imported in vitro into mitochondria isolated from myoclonic epilepsy with ragged-red fiber cells if provided with sufficient ATP (2 mM). This work suggests that tRNA import is more widespread than previously thought and may be a universal trait of mitochondria. Mutations in mitochondrial tRNA genes have been associated with human disease; the tRNA import system described here could possibly be exploited for the manipulation of defective mitochondria.human ͉ MERFF D ifferent protein-synthesizing systems exist in the cytoplasm and organelles (mitochondria and chloroplasts) of eukaryotic cells. Reflecting the evolutionary origin of the organelle, the mitochondrial system is bacteria-like (1, 2). For instance, the tRNAs and aminoacyl-tRNA synthetases present in the mitochondria are closely related to their bacterial counterparts. The mitochondrial genome of higher eukaryotes encodes all of the tRNA species necessary for protein synthesis (3), whereas the aminoacyl-tRNA synthetases are nucleus-encoded and imported into the organelle. The mitochondrial protein-synthesizing system is important for the synthesis of a number of proteins for multienzyme complexes involved in oxidative phosphorylation (1). Since the original suggestion 40 years ago (4), tRNA import into mitochondria has been demonstrated in Tetrahymena, trypanosomatids, yeast, plants, and marsupials (reviewed in refs. 5 and 6). The detailed nature of the different tRNA import mechanisms is not yet known; however, ATP hydrolysis is always required. In the yeast system, the importance of some mitochondrial membrane proteins as well as of specific cytosolic proteins (enolase and lysyl-tRNA synthetase) has been established (reviewed in refs. 5 and 6). After the early observation of the presence of a cytoplasmic tRNA CUU Lys in Saccharomyces cerevisiae mitochondria (7), import o...

All mitochondrial tRNAs in kinetoplastid protists are encoded in the nucleus and imported into the organelle. The tRNA Trp (CCA) can decode the standard UGG tryptophan codon but can not decode the mitochondrial UGA tryptophan codon. We show that the mitochondrial tRNA Trp undergoes a specific C to U nucleotide modification in the first position of the anticodon, which allows decoding of mitochondrial UGA codons as tryptophan. Functional evidence for the absence of a UGA suppressor tRNA in the cytosol, using a reporter gene, was also obtained, which is consistent with a mitochondrial localization of this editing event. Leishmania cells have dealt with the problem of a lack of expression within the organelle of this non-universal tRNA by compartmentalizing an editing activity that modifies the anticodon of the imported tRNA.

Aminoacyl-tRNA (aa-tRNA) formation, an essential process in protein biosynthesis, is generally achieved by direct attachment of an amino acid to tRNA by the aa-tRNA synthetases. An exception is Gln-tRNA synthesis, which in eukaryotes is catalyzed by glutaminyl-tRNA synthetase (GlnRS), while most bacteria, archaea, and chloroplasts employ the transamidation pathway, in which a tRNA-dependent glutamate modification generates Gln-tRNA. Mitochondrial protein synthesis is carried out normally by mitochondrial enzymes and organelle-encoded tRNAs that are different from their cytoplasmic counterparts. Early work suggested that mitochondria use the transamidation pathway for Gln-tRNA formation. We found no biochemical support for this in Saccharomyces cerevisiae mitochondria, but demonstrated the presence of the cytoplasmic GlnRS in the organelle and its involvement in mitochondrial Gln-tRNA synthesis. In addition, we showed in vivo localization of cytoplasmic tRNA Gln in mitochondria and demonstrated its role in mitochondrial translation. We furthermore reconstituted in vitro cytoplasmic tRNA Gln import into mitochondria by a novel mechanism. This tRNA import mechanism expands our knowledge of RNA trafficking in the eukaryotic cell. These findings change our view of the evolution of organellar protein synthesis.

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 af®nity-isolated cytosolic (cy) and mitochondrial (mt) tRNAs for glutamate and glutamine by mass spectrometry. A unique modi®cation difference in both tRNAs was identi®ed 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 modi®cation intermediate for both modi®ed uridines in cy and mt tRNAs. We also isolated a trace amount of mitochondria-speci®c 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 ef®-ciently into isolated mitochondria than the native cy tRNA Glu in an in vitro importation experiment, indicating that cytosol-speci®c 2-thiolation could play an inhibitory role in tRNA import into mitochondria.