The mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episode syndrome-associated human mitochondrial tRNA(Leu(UUR)) mutation causes aminoacylation deficiency and concomitant reduced association of mRNA with ribosomes

The pathogenetic mechanism of the mitochondrial tRNA(Leu(UUR)) A3243G trans ition associated with the mitochondrial myopathy, encephalopathy, lactic ac idosis, and stroke-like episodes (MELAS) syndrome has been investigated in transmitochondrial cell lines constructed by transfer of mutant mitochondri al DNA (mtDNA)-carrying mitochondria from three genetically unrelated MELAS patients or of isogenic wild-type mtDNA-carrying organelles into human mtD NA-less cells. An in vivo footprinting. analysis of the mtDNA segment withi n the tRNA(Leu(UUR)) gene that binds the transcription termination factor f ailed to reveal any difference in occupancy of sites or qualitative interac tion with the protein between mutant and wild-type mtDNAs, Cell lines nearl y homoplasmic for the mutation exhibited a strong (70-75%) reduction in the level of aminoacylated tRNA(Leu(UUR)) and a decrease in mitochondrial prot ein synthesis rate. The latter, however, did not show any significant corre lation between synthesis defect of the individual polypeptides and number o r proportion of UUR codons in their mRNAs, suggesting that another step, ot her than elongation, may be affected. Sedimentation analysis in sucrose gra dient showed a reduction in size of the mitochondrial polysomes, while the distribution of the two rRNA components and of the mRNAs revealed decreased association of mRNA with ribosomes and, in the most affected cell line, pr onounced degradation of the mRNA associated with slowly sedimenting structu res. Therefore, several lines of evidence indicate that the protein synthes is defect in A3243G MELAS mutation-carrying cells is mainly due to a reduce d association of mRNA with ribosomes, possibly as a consequence of the tRNA (Leu(UUR)) amino-acylation defect.