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.

Editing of tRNA has a wide phylogenetic distribution among eukaryotes and in some cases serves to expand the decoding capacity of the target tRNA. We previously described C-to-U editing of the wobble position of the imported tRNA Trp in Leishmania mitochondria, which is essential for decoding UGA codons as tryptophan. Here we show the complete set of nucleotide modifications in the anticodon arm of the mitochondrial and cytosolic tRNA Trp as determined by electrospray ionization mass spectrometry. This analysis revealed extensive mitochondria-specific posttranscriptional modifications, including the first example of thiolation of U33, the "universally unmodified" uridine. In light of the known rigidity imparted on sugar conformation by thiolation, our discovery of a thiolated U33 suggests that conformational flexibility is not a universal feature of the anticodon structural signature. In addition, the in vivo analysis of tRNA Trp variants presented shows a single base-pair reversal in the anticodon stem of tRNA Trp is sufficient to abrogate editing in vivo, indicating that subtle changes in anticodon structure can have drastic effects on editing efficiency.

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 Targeting of one or more nucleus-encoded tRNAs to the mitochondrion occurs in a variety of organisms and allows mitochondrial protein synthesis to proceed in cells in which these tRNAs are not encoded in the mitochondrial genome. Importation of tRNAs into mitochondria has been studied in yeast (1), ciliates (2, 3), plants (4), and trypanosomatids (5-9) using both in vivo and in vitro techniques. In no case is the mechanism of importation of RNA into mitochondria fully understood, and there appear to be differences in the importation process in different organisms. The trypanosomatids Leishmania tarentolae and Trypanosoma brucei represent an extreme situation in which no tRNAs are encoded in the mitochondrial genome; and therefore, all tRNAs for mitochondrial translation must be encoded in the nucleus and imported from the cytosol (10, 11).Targeting of specific tRNAs to the mitochondrion in trypanosomatids has been studied in vivo by transfection techniques (5) and in vitro by importation into isolated mitochondria (12-16). The specificity of targeting as well as the mechanism of transport are still obscure, but the specificity appears to involve, at least in part, the tertiary structure of the RNA molecule (16).It was previously proposed, on the basis of the detection of precursor tRNAs in T. brucei mitochondria, that the substrate for importation is a 5Ј-precursor species that is processed by an RNase P-like activity within the organelle (17). However, Aphasizhev et al. (18) could not obtain any evidence for the existence of such mitochondrion-localized precursors in L. tarentolae or T. brucei. In addition, the presence of a genomic 5Ј-flanking sequence was not required for mitochondrial targeting of tRNA Ile in L. tarentolae (5) or for mitochondrial targeting of tRNA Tyr in T. brucei (8). However, evidence was recently presented that a dicistronic tRNA transcript in T. brucei is the preferred substrate for importation into isolated mitochondria, but no evidence was presented for intramitochondrial processing of this transcript (19,20).To investigate the question of the nature of the in vivo substrate for tRNA importation into the mitochondrion, we have used RT-PCR 1 to analyze the intracellular localization of 5Ј-and 3Ј-end processing and an in vitro assay to analyze the importation properties of several specific tRNAs in L. tarentolae. We have also investigated the subcellular localization of the C to U editing of the anticodon of the tRNA Trp . EXPERIMENTAL PROCEDURESCell Culture, Cell Fractionation, and RNA Isolation-L. tarentolae cells were grown at 27°C in brain/heart infusion medium...

Guide RNAs (gRNAs) are encoded both in the maxicircle and minicircle components of the mitochondrial DNA of trypanosomatid protozoa. These RNAs mediate the precise insertion and deletion of U residues in transcripts of the maxicircle DNA. We showed previously that the old UC laboratory strain of Leishmania tarentolae apparently lost more than 40 minicircle-encoded gRNAs that are present in the recently isolated LEM125 strain [Thiemann et al., EMBO J, 1994, 13:5689-5700]. We have further analyzed the population of minicircle-encoded gRNAs in the LEM125 strain. Sau 3AI and MspI minicircle libraries were constructed and screened for novel gRNAs by negative colony hybridization. This search yielded 20 minicircles encoding new gRNAs that covered most of the remaining gaps in the editing cascades of the ND8, ND9, G4, and G5 genes, and in addition, more than 30 minicircles containing either unassigned or undetectable gRNA genes. We also completely sequenced 34 of the 45 minicircle sequence classes encoding previously identified gRNAs. A total of 19 pairs of redundant gRNAs, which are gRNAs of different sequences covering the same editing blocks, were identified. The gRNAs in each redundant pair generally had different relative abundances and different extents of mismatches with edited sequences. Alignments of the minicircles encoding redundant gRNAs yielded 59 to 93% matching nucleotides, suggesting an origin from duplication of ancestral minicircles and subsequent genetic drift. We propose a functional explanation for the existence of redundant gRNAs in this strain.