In vivo expression and mitochondrial import of normal and mutated tRNA(thr) in Leishmania

Evidence suggests that mitochondria of protozoans and plants contain nuclear-encoded tRNAs. In trypanosomatids, the entire set of tRNAs in the mitochondria are presumably imported from the nucleus, but the mechanism of tRNA import is not presently understood. In this study, we have employed a plasmid-encoded nuclear tRNA gene as a means of investigating tRNA expression and mitochondrial import in vivo in Leishmania tarentolae. Using a Leishmania plasmid, we cloned a 1-kb or 250-bp restriction fragment carrying the nuclear tRNA(thr) gene and three in vitro mutagenized derivatives: Tac6 (an insertion of 6 nucleotides at the anticodon loop), Td4 (a 4-nt insert at the D-loop) and Tv4 (a 4-nt insert at the variable arm). Leishmania cells stably transfected with these plasmids were then examined for tRNA expression and import by Northern analysis. The results show that the plasmid-encoded wild type tRNA(thr) gene produced a significantly elevated level of expression in the cytosol. Similarly, the Tac6-transfected cells exhibited a large abundance of the mutant RNA relative to the normal tRNA (chromosome-encoded gene transcripts) in the cytosol. Furthermore, the mutant Tac6 RNA was found imported into mitochondria, although the proportion of the mutant vs. normal tRNA in mitochondria was greatly reduced as compared to that in the cytosol. We suggest that the mitochondrial import machinery is capable of discriminating against the mutant RNA in favor of the normal tRNA for import. In another example, we found that the Tv4 gene showed expression, albeit somewhat reduced, but its import into mitochondria was completely blocked. Unexpectedly, the 4-base addition mutation (Td4) at the D-loop showed neither expression nor import. While these results clearly signify the importance of various segments within the tRNA gene for in vivo expression, our data underscore the significance of the variable loop for mitochondrial import. It is our belief that this plasmid-encoded tRNA gene expression system in Leishmania may be useful in gaining further insights on tRNA import.