The Mitochondrial Myopathy, Encephalopathy, Lactic Acidosis, and Stroke-like Episode Syndrome-associated Human Mitochondrial tRNALeu(UUR) Mutation Causes Aminoacylation Deficiency and Concomitant Reduced Association of mRNA with Ribosomes

Abstract

The pathogenetic mechanism of the mitochondrial tRNA Leu(UUR) A3243G transition associated with the mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome has been investigated in transmitochondrial cell lines constructed by transfer of mutant mitochondrial DNA (mtDNA)-carrying mitochondria from three genetically unrelated MELAS patients or of isogenic wild-type mtDNA-carrying organelles into human mtDNA-less cells. An in vivo footprinting analysis of the mtDNA segment within the tRNA Leu(UUR) gene that binds the transcription termination factor failed to reveal any difference in occupancy of sites or qualitative interaction with the protein between mutant and wild-type mtDNAs. Cell lines nearly homoplasmic for the mutation exhibited a strong (70 -75%) reduction in the level of aminoacylated tRNA Leu(UUR) and a decrease in mitochondrial protein synthesis rate. The latter, however, did not show any significant correlation between synthesis defect of the individual polypeptides and number or proportion of UUR codons in their mRNAs, suggesting that another step, other than elongation, may be affected. Sedimentation analysis in sucrose gradient 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, pronounced degradation of the mRNA associated with slowly sedimenting structures. Therefore, several lines of evidence indicate that the protein synthesis defect in A3243G MELAS mutation-carrying cells is mainly due to a reduced association of mRNA with ribosomes, possibly as a consequence of the tRNA Leu(UUR) aminoacylation defect.

Codon-specific translational defect caused by a wobble modification deficiency in mutant tRNA from a human mitochondrial disease

“…These dysfunctions include impaired termination (34), impaired pre-tRNA processing (35), decreased stability and aminoacylation (36), and abnormal conformation (37), and commonly lead to a decreased steady-state level of the normal aminoacylated tRNA, which in turn leads to reduced protein synthesis. Some of these biochemical tRNA analyses were performed by using an unmodified tRNA transcribed in vitro, because it is difficult to obtain a sufficient number of native mt tRNAs with the MELAS mutation for a series of analyses.…”

Aminoacylation properties of pathology-related human mitochondrial tRNALysvariants

“…On the other hand, two mutations linked to encephalopathies do affect this reaction (G8313A and G8328A), whereas a third one (T8316C, linked to MELAS) does not. MELAS syndrome correlates with mutation A3243G in tRNA Leu(UUR) , in which it leads to a significant decrease in leucylation (see Börner et al 2000;Chomyn et al 2000;Park et al 2003;Sohm et al 2003). Thus, primary mechanisms leading to the same disorder can be different according to the tRNA in which the deleterious mutation occurs.…”

Overexpressed mitochondrial leucyl-tRNA synthetase suppresses the A3243G mutation in the mitochondrial tRNALeu(UUR) gene

“…The mutation does not alter the accuracy of processing of the mtDNA polycistronic RNA transcript at tRNA Leu(UUR) (King et al 1992;Koga et al 1993;Kaufmann et al 1996). A3243G mutant cells have decreased steady-state levels of tRNA Leu(UUR) (Chomyn et al 1992;Janssen et al 1999;Chomyn et al 2000;Yasukawa et al 2000;Park et al 2003) and can also have reductions in the fraction of tRNA Leu(UUR) that is aminoacylated (El Meziane et al 1998;Janssen et al 1999;Borner et al 2000;Chomyn et al 2000;Park et al 2003). The reduction in the fraction of aminoacylated tRNA Leu(UUR) is likely due to a 25-fold decrease in aminoacylation efficiency of A3243G mutant tRNA Leu(UUR) as compared with wild-type tRNA Leu(UUR) (Park et al 2003).…”