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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 (tRNATrp) is edited by a C to U modification at the first position of the anticodon. To determine the in vivosubstrates for mitochondrial tRNA importation as well as tRNA editing, we examined the subcellular localization and extent of 5′- and 3′-end maturation of tRNATrp(CCA), tRNAIle(UAU), tRNAGln(CUG), tRNALys(UUU), and tRNAVal(CAC). Nuclear, cytosolic, and mitochondrial fractions were obtained with little cross-contamination, as determined by Northern analysis of specific marker RNAs. tRNAGln was mainly cytosolic in localization; tRNAIle and tRNALys were mainly mitochondrial; and tRNATrpand tRNAVal were shared between the two compartments. 5′- and 3′-extended precursors of all five tRNAs were present only in the nuclear fraction, suggesting that the mature tRNAs represent thein vivo substrates for importation into the mitochondrion. Consistent with this model, T7-transcribed mature tRNAIleunderwent importation in vitro into isolated mitochondria more efficiently than 5′-extended precursor tRNAIle. 5′-Extended precursor tRNATrp was found to be unedited, which is consistent with a mitochondrial localization of this editing reaction. T7-transcribed unedited tRNATrp was imported in vitro more efficiently than edited tRNATrp, suggesting the presence of importation determinants in the anticodon.