Biogenesis of glutaminyl-mt tRNA ^Gln in human mitochondria

Abstract

Mammalian mitochondrial (mt) tRNAs, which are required for mitochondrial protein synthesis, are all encoded in the mitochondrial genome, while mt aminoacyl-tRNA synthetases (aaRSs) are encoded in the nuclear genome. However, no mitochondrial homolog of glutaminyl-tRNA synthetase (GlnRS) has been identified in mammalian genomes, implying that Gln-tRNA Gln is synthesized via an indirect pathway in the mammalian mitochondria. We demonstrate here that human mt glutamyl-tRNA synthetase (mtGluRS) efficiently misaminoacylates mt tRNA Gln to form Glu-tRNA Gln . In addition, we have identified a human homolog of the Glu-tRNA Gln amidotransferase, the hGatCAB heterotrimer. When any of the hGatCAB subunits were inactivated by siRNA-mediated knock down in human cells, the Glu-charged form of tRNA Gln accumulated and defects in respiration could be observed. We successfully reconstituted in vitro Gln-tRNA Gln formation catalyzed by the recombinant mtGluRS and hGatCAB. The misaminoacylated form of tRNA Gln has a weak binding affinity to the mt elongation factor Tu (mtEF-Tu), indicating that the misaminoacylated form of tRNA Gln is rejected from the translational apparatus to maintain the accuracy of mitochondrial protein synthesis.EF-Tu ͉ Glu-tRNAGln amidotransferase ͉ glutamyl-tRNA synthetase ͉ mitochondrial tRNA

Mutations in the mitochondrial cysteinyl-tRNA synthase gene,CARS2,lead to a severe epileptic encephalopathy and complex movement disorder

“…To facilitate their import, nuclear-encoded ARSs carry a mitochondrial targeting sequence within the mitochondrial ARSs (mt-ARSs) polypeptide. In total, 17 of the 37 nuclear-encoded ARSs genes including CARS2 are exclusively mitochondrial targeted, two are bi-functional in cytoplasm and mitochondria, whereas one of the mt-tRNA (Gln) is aminoacylated via an indirect pathway 32. Thus, the mutations in CARS2 detected in our patient, both of which are predicted to be deleterious, are likely to lead to defects in mitochondrial translation.…”

Trmt61B is a methyltransferase responsible for 1-methyladenosine at position 58 of human mitochondrial tRNAs

“…In mammalian mitochondria, all 22 tRNA species are encoded in mitochondrial DNA (Anderson et al 1981 (Yokogawa et al 1991;Yasukawa et al 2000;Nagao et al 2009). To date, the functional role and biogenesis of m 1 A58 in mammalian mitochondrial tRNAs remain to be elucidated (Suzuki et al 2011).…”

Characterization of Gtf1p, the Connector Subunit of Yeast Mitochondrial tRNA-dependent Amidotransferase

“…is known to occur in prokaryotes and more recently has also been shown in eukaryotic organelles (3,5,6). In bacteria, the AdT reactions are catalyzed by the heterotrimeric enzyme encoded by the GatCAB operon (7,8).…”