Dual targeting of a single tRNA ^Trp requires two different tryptophanyl-tRNA synthetases in Trypanosoma brucei

Abstract

The mitochondrion of Trypanosoma brucei does not encode any tRNAs. This deficiency is compensated for by the import of a small fraction of nearly all of its cytosolic tRNAs. Most trypanosomal aminoacyl-tRNA synthetases are encoded by single-copy genes, suggesting the use of the same enzyme in the cytosol and mitochondrion. However, the T. brucei genome contains two distinct genes for eukaryotic tryptophanyl-tRNA synthetase (TrpRS). RNA interference analysis established that both TrpRS1 and TrpRS2 are essential for growth and required for cytosolic and mitochondrial tryptophanyl-tRNA formation, respectively. Decoding the mitochondrial tryptophan codon UGA requires mitochondria-specific C3 U RNA editing in the anticodon of the imported tRNA Trp . In vitro charging assays with recombinant TrpRS enzymes demonstrated that the edited anticodon and the mitochondria-specific thiolation of U33 in the imported tRNA Trp act as antideterminants for the cytosolic TrpRS1. The existence of two TrpRS enzymes, therefore, can be explained by the need for a mitochondrial synthetase with extended substrate specificity to achieve aminoacylation of the imported thiolated and edited tRNA Trp . Thus, the notion that, in an organism, all nuclear-encoded tRNAs assigned to a given amino acid are charged by a single aminoacyl-tRNA synthetase, is not universally valid.genetic code ͉ mitochondria ͉ RNA editing ͉ aminoacyl-tRNA synthetase

A mechanism for functional segregation of mitochondrial and cytosolic genetic codes

“…TbKRS1 and TbKRS2 expressing cell lines were fractionated using digitonin extraction protocol described elsewhere (13). Thirty thousand cell equivalents per fraction were electrophoresed and analyzed by immunoblot using BB2 antibody.…”

“…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). Differences between mitochondrial and cytosolic ribosomes may be responsible for the existence of these differentially modified tRNAs (15).…”

Thiolated tRNAs of Trypanosoma brucei Are Imported into Mitochondria and Dethiolated after Import

“…tarentolae and T. brucei are closely related, and most aspects of mitochondrial tRNA import that have been investigated in the two species are highly similar. Both lack mitochondrial tRNA genes (37,38), show identical import specificity and edit the imported tRNA Trp to accommodate the variant genetic code (11,12). Whether the actual tRNA import machinery is also identical is unclear at present.…”

“…Moreover, analysis of digitonin-extracted mitochondrial fractions showed that the tagged tRNA Glu (UUC) is thiolated and imported into mitochondria. (12). The top panel shows that the cytosolic and mitochondrial RNA fractions that were used as templates for RT are free of DNA.…”

“…It has been shown that the cytidine 32 modifications are not required for import of the affected tRNAs but rather are a consequence of the mitochondrial localization of the tRNAs (9). Moreover, C to U editing of the imported tRNA Trp is required for correct decoding of the UGA codon, because in trypanosomatid mitochondria this codon has been re-assigned to tryptophan (11,12). Finally, it has been proposed that in L. tarentolae the cytosol-specific thio modifications of the tRNA Glu (UUC) and tRNA Gln (UUG) might be antideterminants for mitochondrial import (13).…”