End processing precedes mitochondrial importation and editing of tRNAs in Leishmania tarentolae.
Kapushoc ST, Alfonzo JD, Rubio MA, Simpson L.
Departments
of Molecular, Cell, and Developmental Biology and Microbiology,
Immunology, and Molecular Genetics and the Howard Hughes Medical
Institute, University of California, Los Angeles, California 90095, USA.
Abstract
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 of the anticodon. To determine the in vivo substrates for
mitochondrial tRNA importation as well as tRNA editing, we examined the
subcellular localization and extent of 5'- and 3'-end maturation of
tRNA(Trp)(CCA), tRNA(Ile)(UAU), 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 mitochondrial; and
tRNA(Trp) and tRNA(Val) 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 the in vivo
substrates for importation into the mitochondrion. Consistent with this
model, T7-transcribed mature tRNA(Ile) underwent importation in vitro
into isolated mitochondria more efficiently than 5'-extended precursor
tRNA(Ile). 5'-Extended precursor tRNA(Trp) was found to be unedited,
which is consistent with a mitochondrial localization of this editing
reaction. T7-transcribed unedited tRNA(Trp) was imported in vitro more
efficiently than edited tRNA(Trp), suggesting the presence of
importation determinants in the anticodon.
