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.