In vivo import of unspliced tRNA(Tyr) containing synthetic introns of variable length into mitochondria of Leishmania tarentolae

Abstract

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 tRNA(Tyr), which in trypanosomatids contains an 11 nt intron. Previous work has defined an exon mutation which leads to accumulation of unspliced precursor tRNA(Tyr). In this study we have used the splicing-deficient tRNA(Tyr) 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 tRNA(Tyr) precursors were expressed in transgenic cell lines. Whereas stable expression of precursor tRNAs(Tyr) was obtained for introns up to a length of 76 nt, only precursors having introns up to 38 nt were imparted into mitochondria. These results demonstrate that splicing-deficient tRNA(Tyr) 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 molecule.