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.

PCR amplification of template DNAs extracted from mixed, naturally occurring microbial populations, using oligonucleotide primers complementary to highly conserved sequences, was used to obtain a large collection of diverse RNase P RNA-encoding genes. An (Fig. 1). Some new RNase P RNA gene sequences arose as contaminants ("volunteer" sequences) in PCRs using known template DNAs. Although of unknown origin, they are authentic RNase P RNAs based on similarity to known RNase P RNAs and proved useful in the structure analysis. PCRs were performed and product DNAs were cloned essentially as described (2). Fragments containing "70% of each of the RNase P RNA-encoding genes were amplified by using oligonucleotide primers 59FBam (5'-CGGGATCCGIIGAG-GAAAGTCCIIGC-3'; I = inosine) and 347REco (5'-CGGAATTCRTAAGCCGGRTTCTGT-3'; R = A or G) and separated by preparative electrophoresis in 3% agarose gels (NuSieve GTG, FMC BioProducts) after digestion with restriction endonucleases EcoRI and BamHI. The diffuse band corresponding to DNA amplification products of =300 bp was excised from the gel, ligated into EcoRI/BamHI-digested pBluescript KS+ DNA (Stratagene, Inc.), and transformed into Escherichia coli DH5aF'. Double-stranded plasmid DNAs were sequenced by the dideoxynucleotide chain-termination method using Sequenase version 2.0 (United States Biochemicals) (10). Clones containing unique RNase P RNA sequences based on sequence data from a single primer were completely sequenced on both strands using M13 universal and reverse primers, 59FBam, 347REco, 174F (5'-AGGGTGAAANGGTGSGGTAAGAG-3'; N = A, Abbreviations: To denote interactions between bases we use a slash for canonical base pairs (e.g., G/C and A/U), a dot for noncanonical interactions (e.g., G-U and A.G), and a colon for a triple interaction between a base and a base pair (e.g., A:G/C); Nomenclature of structural elements in RNase P RNA corresponds to the group I intron convention (1), as described (2): P (paired) refers to a helix, numbered according to encounter from the 5' end; L refers to the loop of particular helices; and J (joining) refers to the nucleotide stretch between particular helices (e.g., J5/6 connects P5 and P6). SPresent address:

Transfer RNA modifications play pivotal roles in protein synthesis. N6-threonylcarbamoyladenosine (t6A) and its derivatives are modifications found at position 37, 3΄-adjacent to the anticodon, in tRNAs responsible for ANN codons. These modifications are universally conserved in all domains of life. t6A and its derivatives have pleiotropic functions in protein synthesis including aminoacylation, decoding and translocation. We previously discovered a cyclic form of t6A (ct6A) as a chemically labile derivative of t6A in tRNAs from bacteria, fungi, plants and protists. Here, we report 2-methylthio cyclic t6A (ms2ct6A), a novel derivative of ct6A found in tRNAs from Bacillus subtilis, plants and Trypanosoma brucei. In B. subtilis and T. brucei, ms2ct6A disappeared and remained to be ms2t6A and ct6A by depletion of tcdA and mtaB homologs, respectively, demonstrating that TcdA and MtaB are responsible for biogenesis of ms2ct6A.

Nucleic acids undergo naturally occurring chemical modifications. Over 100 different modifications have been described and every position in the purine and pyrimidine bases can be modified; often the sugar is also modified1. Despite recent progress, the mechanism for the biosynthesis of most modifications is not fully understood, owing, in part, to the difficulty associated with reconstituting enzyme activity in vitro. Whereas some modifications can be efficiently formed with purified components, others may require more intricate pathways2. A model for modification interdependence, in which one modification is a prerequisite for another, potentially explains a major hindrance in reconstituting enzymatic activity in vitro3. This model was prompted by the earlier discovery of tRNA cytosine-to-uridine editing in eukaryotes, a reaction that has not been recapitulated in vitro and the mechanism of which remains unknown. Here we show that cytosine 32 in the anticodon loop of Trypanosoma brucei tRNAThr is methylated to 3-methylcytosine (m3C) as a pre-requisite for C-to-U deamination. Formation of m3C in vitro requires the presence of both the T. brucei m3C methyltransferase TRM140 and the deaminase ADAT2/3. Once formed, m3C is deaminated to 3-methyluridine (m3U) by the same set of enzymes. ADAT2/3 is a highly mutagenic enzyme4, but we also show that when co-expressed with the methyltransferase its mutagenicity is kept in check. This helps to explain how T. brucei escapes ‘wholesale deamination’5 of its genome while harbouring both enzymes in the nucleus. This observation has implications for the control of another mutagenic deaminase, human AID, and provides a rationale for its regulation.