C to U editing of the anticodon of imported mitochondrial tRNATrp allows decoding of the UGA stop codon in Leishmania tarentolae

Abstract

All mitochondrial tRNAs in kinetoplastid protists are encoded in the nucleus and imported into the organelle. The tRNA Trp (CCA) can decode the standard UGG tryptophan codon but can not decode the mitochondrial UGA tryptophan codon. We show that the mitochondrial tRNA Trp undergoes a specific C to U nucleotide modification in the first position of the anticodon, which allows decoding of mitochondrial UGA codons as tryptophan. Functional evidence for the absence of a UGA suppressor tRNA in the cytosol, using a reporter gene, was also obtained, which is consistent with a mitochondrial localization of this editing event. Leishmania cells have dealt with the problem of a lack of expression within the organelle of this non-universal tRNA by compartmentalizing an editing activity that modifies the anticodon of the imported tRNA.

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

“…Thus, it is unexpected that trypanosomatids import the tRNA Trp . The way Leishmania mitochondria accommodate the change in the genetic code is by mitochondria-specific C3U editing of the first anticodon position of the imported tRNA Trp (13). In the present work, we have analyzed the situation in the closely related T. brucei and shown that although editing of the imported tRNA Trp is necessary, it is not sufficient to allow decoding of the mitochondrial UGA codons.…”

“…In most systems, the reassignment of the UGA codon is accommodated by a single mutation in the mitochondrial tRNA Trp gene, which probably requires an adaptation of the mitochondrial TrpRS (3). The situation is different for mitochondria of trypanosomatids because they need (i) a mitochondria-specific tRNA editing enzyme that produces the tRNA Trp UCA (13) and (ii) a separate TrpRS (Tb-TrpRS2) with an extended substrate specificity. The need for a record TrpRS is due to the fact that the C3U editing not only changes the decoding properties of the tRNA Trp but also its identity toward the classic TrpRS (Tb-TrpRS1).…”

“…However, the only tRNA Trp gene in T. brucei is nuclear and carries the standard tryptophan anticodon CCA. Recent work in Leishmania revealed that trypanosomatids use RNA editing to convert the CCA anticodon of Ϸ40% of the imported tRNA Trp to UCA; the resulting tRNA now is able to recognize UGA codons (13). Besides RNA editing, the imported tRNA Trp is subjected to additional mitochondria-specific modifications; most importantly, the thiolation of U33, the ''universally unmodified'' uridine in all known tRNAs (14).…”

“…It is not obvious why T. brucei should have two aspartyl-or lysyl-tRNA synthetases, because the corresponding cytosolic and mitochondrial substrate tRNAs derived from the same nuclear genes. However, the need for two distinct TrpRSs might be explained by the fact that because of editing in the mitochondrion, cytosolic and mitochondrial tRNA Trp differ in an anticodon nucleotide (13). In this study, we focus on the functional analysis of Tb-TrpRS1 and Tb-TrpRS2, the two trypanosomal TrpRS homologues.…”

From simple to supercomplex: mitochondrial genomes of euglenozoan protists

“…The gRNAs provide information for post-transcriptional RNA editing in the form of multiple insertions and deletions of uridine residues into the maxicircle-derived mRNAs (for recent reviews, see 19, 27, 28). The kDNA does not encode tRNA genes, and hence all tRNA molecules must be imported from the cytosol 29, 30 .…”