Dual targeting of isoleucyl-tRNA synthetase in Trypanosoma brucei is mediated through alternative trans-splicing

Abstract

Aminoacyl-tRNA synthetases catalyze the aminoacylation of tRNAs with their cognate amino acids. They are an essential part of each translation system and in eukaryotes are therefore found in both the cytosol and mitochondria. Thus, eukaryotes either have two distinct genes encoding the cytosolic and mitochondrial isoforms of each of these enzymes or a single gene encoding dually localized products. Trypanosomes require trans-splicing of a cap containing leader sequence onto the 5′-untranslated region of every mRNA. Recently we speculated that alternative trans-splicing could lead to the expression of proteins having amino-termini of different lengths that derive from the same gene. We now demonstrate that alternative trans-splicing, creating a long and a short spliced variant, is the mechanism for dual localization of trypanosomal isoleucyl-tRNA synthetase (IleRS). The protein product of the longer spliced variant possesses an amino-terminal presequence and is found exclusively in mitochondria. In contrast, the shorter spliced variant is translated to a cytosol-specific isoform lacking the presequence. Furthermore, we show that RNA stability is one mechanism determining the differential abundance of the two spliced isoforms.

A Multiple Aminoacyl-tRNA Synthetase Complex That Enhances tRNA-Aminoacylation in African Trypanosomes

“…Most of the annotated aaRSs had been identified in the mitochondrial proteome of T. brucei procyclic forms (38). Five of the dually localized aaRSs are encoded by alternatively trans-spliced transcripts (45,46), which provides for the joint localization, as reported by others (29,30) and confirmed Table 6. Data in panel g are the averages of three experiments Ϯ SDs, and those in panel f are the averages of four experiments Ϯ SDs.…”

“…Alternative mRNA transsplicing has previously been shown for other mRNAs in T. brucei, i.e., those which encode IleRS, ProRS, GluRS, GlnRS, and asparaginyl-tRNA synthetase (AsnRS) (45,46). This results in two mRNA isoforms that either retain or lack the N-terminal mitochondrial targeting sequence (MTS), and dual cytoplasmic and mitochondrial localization was confirmed for IleRS in procyclic forms (29). We expressed the spliced variants of IleRS, ProRS, and GluRS with a 3V5 tag in bloodstream forms and showed dual cytoplasmic and mitochondrial localization by immunofluorescence staining (Fig.…”

“…and T. cruzi. Few aaRSs have been characterized in T. brucei, and most of the genetic studies have been conducted on the insect stage of the parasite (24)(25)(26)(27)(28)(29)(30)(31). Although in T. brucei translation takes place in both the cytoplasm and the mitochondria, only 24 genes have been identified in the genome as potentially encoding aaRSs (32).…”

“…Although in T. brucei translation takes place in both the cytoplasm and the mitochondria, only 24 genes have been identified in the genome as potentially encoding aaRSs (32). A few of these genes have been shown to encode both cytoplasmic and mitochondrial enzymes (25)(26)(27)30), and dual cytoplasmic and mitochondrial localization has been shown to result from alternative trans-splicing of IleRS mRNA (29). How these parasites aminoacylate all the tRNAs that are needed for cytoplasmic and mitochondrial translation with this limited set of genes remains to be determined.…”

Genetic Validation of Aminoacyl-tRNA Synthetases as Drug Targets in Trypanosoma brucei

“…Almost all aaRSs in the T. brucei genome are single copy and thus must serve dual roles in the cytosol and mitochondria; TrpRS, LysRS, and AspRS are three notable exceptions where separate cytosolic and mitochondrial genes have been identified (24)(25)(26). Several T. brucei aaRS genes have been reported to be essential in parasites in either the insect stage (procyclic) or the mammalian stage (21,(25)(26)(27)(28)(29)(30). Several groups have also reported the identification of MetRS, IleRS, and LeuRS inhibitors with antitrypanosome activity (30)(31)(32)(33)(34)(35).…”