End processing precedes mitochondrial importation and editing of tRNAs in Leishmania tarentolae

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 o f the anticodon, To determine the in vivo substrates for mitochondrial tRNA importation as well as tRNA editing, we examined the subcellular localizat ion and extent of 5'- and 3'-end maturation of tRNA(Trp)(CCA), tRNA(Ile)(UA U), tRNA(Gln)(CUG), tRNA(Lys)(UUU), and tRNA(Val)(CAC), Nuclear, cytosolic, and mitochondrial fractions were obtained with little cross-contamination, as determined by Northern analysis of specific marker RNAs, tRNA(Gln) was mainly cytosolic in localization; tRNA(Ile) and tRNA(Lys) were mainly mitoc hondrial; and tRNA(Trp) and tRNA(Val) were shared between the two compartme nts. 5'- and 3'-extended precursors of all five tRNAs were present only in the nuclear fraction, suggesting that the mature tRNAs represent the in viv o substrates for importation into the mitochondrion, Consistent with this m odel, T7-transcribed mature tRNA(Ile) underwent importation in vitro into i solated mitochondria more efficiently than 5'-extended precursor tRNA(Ile). 5'-Extended precursor tRNA(Trp) was found to be unedited, which is consist ent with a mitochondrial localization of this editing reaction, T7-transcri bed unedited tRNA(Trp) was imported in vitro more efficiently than edited t RNA(Trp), suggesting the presence of importation determinants in the antico don.