tRNAs are imported into mitochondria of Trypanosoma brucei independently of their genomic context and genetic origin.

Abstract

The mitochondrial genome of Trypanosoma brucei does not encode any identifiable tRNAs. Instead, mitochondrial tRNAs are synthesized in the nucleus and subsequently imported into mitochondria. In order to analyse the signals which target the tRNAs into the mitochondria, an in vivo import system has been developed: tRNA variants were expressed episomally and their import into mitochondria assessed by purification and nuclease treatment of the mitochondrial fraction. Three tRNA genes were tested in this system: (i) a mutated version of the trypanosomal tRNA(Tyr); (ii) a cytosolic tRNA(His) of yeast; and (iii) a human cytosolic tRNA(Lys). The tRNAs were expressed in their own genomic context, or containing various lengths of the 5′‐flanking sequence of the trypanosomal tRNA(Tyr) gene. In all cases efficient import of each of the tRNAs was observed. We independently confirmed the mitochondrial import of the yeast tRNA(His), since in organello [alpha‐32P]ATP‐labelling of the 3′‐end of the tRNA was inhibited by carboxyatractyloside, a highly specific inhibitor of the mitochondrial adenine nucleotide translocator. Import of heterologous tRNAs in their own genomic contexts supports the conclusion that no specific targeting signals are necessary to import tRNAs into mitochondria of T. brucei, but rather that the tRNA structure itself is sufficient to specify import.

The anticodon is the signal sequence for mitochondrial import of glutamine tRNA in Tetrahymena.

“…Evidence that specific RNA import in trypanosomatids is achieved by the presence of antideterminants within nonimported tRNAs is less direct, but intriguing. In Trypanosoma brucei, tRNAs from yeast and human as well as mutants of T. bruceiimported tRNAs all exhibit some mitochondrial localization when expressed from nuclear genes (Hauser and Schneider 1995). This led to the conclusion that no specific signals were required to achieve tRNA import in this organism, but that the tRNA structure alone was sufficient to direct import (Hauser and Schneider 1995), a conclusion consistent with the proposed idea.…”

“…Therefore, the conclusions drawn from these data regarding the role of synthetases in tRNA import may not be generalizable to the import of multiple tRNAs for use in mitochondrial translation. Perhaps more relevant to the general question of the role of synthetases in the import of tRNAs is recent work in T. brucei (Schneider et al 1994;Hauser and Schneider 1995). Using an in vivo tRNA import assay, these workers found that a mutant tRNA Tyr containing an unsplicable intron could be imported into mitochondria.…”

Eukaryotic-type elongator tRNA Met of Trypanosoma brucei becomes formylated after import into mitochondria

“…Northern blots were done as described (17). The indicated oligonucleotides directed against the T-stem loop region were used for the specific detection of tRNA Met-i (GT-TGGTTTCGATCCAACG) and tRNA Met-e (GTGAGGCTC-GAACTCACG).…”

A mechanism for functional segregation of mitochondrial and cytosolic genetic codes

“…It is unclear why all eukaryotic cells maintain separate translational apparatuses for the cytosol and the mitochondria. This situation is particularly intriguing among kinetoplastids, where only nuclear genes code for tRNAs which function indistinctively in the cytosol and in the mitochondria, but mitochondria-specific ARS still exist (12). It has been determined that at least some of the Trypanosoma and Leishmania tRNAs that are transported to the mitochondria become modified (13,14).…”