Identification of nuclear encoded precursor tRNAs within the mitochondrion of Trypanosoma brucei

RNAs that function in mitochondria are typically encoded by the mitochondrial DNA. However, the mitochondrial tRNAs of Trypanosoma brucei are encoded by the nuclear DNA and therefore must be imported into the mitochondrion. It is becoming evident that RNA import into mitochondria is phylogenetically widespread and is essential for cellular processes, but virtually nothing is known about the mechanism of RNA import. We have identified and characterized mitochondrial precursor tRNAs in T. brucei. The identification of mitochondrially located precursor tRNAs clearly indicates that mitochondrial tRNAs are imported as precursors. The mitochondrial precursor tRNAs hybridize to cloned nuclear tRNA genes, label with [alpha-32P]CTP using yeast tRNA nucleotidyltransferase and in isolated mitochondria via an endogenous nucleotidyltransferase-like activity, and are processed to mature tRNAs by Escherichia coli and yeast mitochondrial RNase P. We show that T. brucei mitochondrial extract contains an RNase P activity capable of processing a prokaryotic tRNA precursor as well as the T. brucei tRNA precursors. Precursors for tRNA(Asn) and tRNA(Leu) were detected on Northern blots of mitochondrial RNA, and the 5' ends of these RNAs were characterized by primer extension analysis. The structure of the precursor tRNAs and the significance of nuclear encoded precursor tRNAs within the mitochondrion are discussed.