All of the mitochondrial tRNAs of Trypanosoma brucei have been shown to be encoded in the nucleus and must be imported into the mitochondrion. The import of nuclearly encoded tRNAs into the mitochondrion has been demonstrated in a variety of organisms and is essential for proper function in the mitochondrion. An in vitro import assay has been developed to study the pathway of tRNA import in T. brucei. The in vitro system utilizes crude isolated trypanosome mitochondria and synthetic RNAs transcribed from a cloned nucleus-encoded tRNA gene cluster. The substrate, composed of tRNA(Ser) and tRNA(Leu), is transcribed in tandem with a 59-nucleotide intergenic region. The tandem tRNA substrate is imported rapidly, while the mature-size tRNA(Leu) fails to be imported in this system. These results suggest that the preferred substrate for tRNA import into trypanosome mitochondria is a precursor molecule composed of tandemly linked tRNAs. Import of the tandem tRNA substrate requires (i) a protein component that is associated with the surface of the mitochondrion, (ii) ATP pools both outside and within the mitochondrion, and (iii) a membrane potential. Dissipation of the proton gradient across the inner mitochondrial membrane by treatment with an uncoupling agent inhibits import of the tandem tRNA substrate. Characterization of the import requirements indicates that mitochondrial RNA import proceeds by a pathway including a protein component associated with the outer mitochondrial membrane, ATP-dependent steps, and a mitochondrial membrane potential.
The mitochondrial tRNAs of Trypanosoma brucei are nuclear encoded and imported into the mitochondrion. A heterogeneous population of RNAs having characteristics of precursor tRNAs have previously been identified within the mitochondrion of T. brucei, suggesting that import occurs via a precursor molecule. In order to identify nuclear genes encoding tRNAs targeted to the mitochondrion, individual mitochondrial tRNAs were separated using two-dimensional gel electrophoresis and enzymatically sequenced. A 1.1-kilobase pair genomic DNA fragment was cloned containing three tRNA genes, tRNA 1 Ser , tRNA Leu , and tRNA 2 Ser . Dicistronic precursors containing the tRNA 1Ser and tRNA Leu transcripts with a 59-nucleotide intergenic sequence were identified by reverse transcriptase and polymerase chain reactions and the 5 end of the precursors determined. The dicistronic precursor tRNA is present both in the cytosol and the mitochondrion supporting a model for tRNA import involving precursor tRNA transcripts.
Trypanosoma brucei lacks mitochondrial genes encoding tRNAs and must import nuclearly encoded tRNAs from the cytosol. The mechanism and specificity of this process remain unclear. We have identified a unique sequence motif, YGG(C/A)RRC, upstream of the genes encoding mitochondrially localized tRNAs in T. brucei. Both in vitro import studies and in vivo transfection studies indicate that deletion of the YGG(C/A)RRC sequence alters mitochondrial localization of tRNA Leu , and in vivo studies also show a decrease in the cellular abundance of tRNA Leu . These studies provide direct evidence for cis-acting RNA motifs within precursor tRNAs that facilitate the selection of tRNAs for mitochondrial import in trypanosomes. Furthermore, we found that mutations to the YGG(C/A)RRC sequence also altered the intracellular distribution of other endogenous tRNAs, suggesting a general role for this sequence in tRNA trafficking in trypanosomes.
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