In vivo import of unspliced tRNATyr containing synthetic introns of variable length into mitochondria of Leishmania tarentolae

The mitochondrial genomes of trypanosomatids lack tRNA genes. Instead, mitochondrial tRNAs are encoded and synthesized in the nucleus and are then imported into mitochondria. This also applies for tRNATyr, which in trypanosomatids contains an 11 nt intron. Previous work has defined an exon mutation which leads to accumulation of unspliced precursor tRNATyr. In this study we have used the splicing-deficient tRNATyr as a vehicle to introduce foreign sequences into the mitochondrion of Leishmania tarentolae. The naturally occurring intron was replaced by synthetic sequences of increasing length and the resulting tRNATyr precursors were expressed in transgenic cell lines. Whereas stable expression of precursor tRNAsTyr was obtained for introns up to a length of 76 nt, only precursors having introns up to 38 nt were imported into mitochondria. These results demonstrate that splicing-deficient tRNATyr can be used to introduce short synthetic sequences into mitochondria in vivo. In addition, our results show that one factor which limits the efficiency of import is the length of the molecul