Human Molecular Genetics Advance Access published online on August 27, 2008
Human Molecular Genetics, doi:10.1093/hmg/ddn265
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The A3243G tRNALeu(UUR) MELAS mutation causes amino acid misincorporation and a combined respiratory chain assembly defect that is partially suppressed by overexpression of the translation elongation factors EFTu and EFG2
1 Montreal Neurological Institute, Montreal, H3A 2B4, Canada 2 Department of Human Genetics, McGill University, Montreal, H3A 2B4, Canada
* Correspondence to: Dr. Eric A. Shoubridge, Montreal Neurological Institute, 3801 University Street, Montreal, Quebec, H3A 2B4, Canada Tel.: +514 398 8523, Fax: +514 398 1509, E-mail: eric{at}ericpc.mni.mcgill.ca
Received June 10, 2008; Revised August 22, 2008; Accepted August 22, 2008
The majority of patients with MELAS (mitochondrial encephalomyophathy, lactic acidosis, stroke-like episodes) carry a heteroplasmic A3243G mutation in the mitochondrial tRNALeu(UUR). The mutation prevents modification of the wobble U base, impairing translation at UUA and UUG codons; however, whether this results in amino acid misincorporation in the mitochondrial translation products remains controversial. We tested this hypothesis in homoplasmic mutant myoblasts isolated from a MELAS patient and investigated whether overexpression of the mitochondrial translation elongation factors could suppress the translation defect. Blue-Native gel electrophoretic analysis demonstrated an almost complete lack of assembly of respiratory chain complexes I, IV and V in MELAS myoblasts. This phenotype could be partially suppressed by overexpression of EFTu or EFG2, but not EFTs or EFG1. Despite the severity of the assembly defect, overall mitochondrial protein synthesis was only moderately affected, but some anomalously migrating translation products were present. Pulse-chase labeling showed reduced stability of all mitochondrial translation products consistent with the assembly defect. Labeling patterns of the translation products were similar with [3H]-leucine or [3H]-phenylalanine, showing that loss of the wobble U modification did not permit decoding of UUY codons; however, endoproteinase fingerprint analysis showed clear evidence of amino acid misincorporation in three polypeptides: CO III, CO II and ATP6. Taken together, these data demonstrate that the A3243G mutation produces both loss- and gain-of function phenotypes, explaining the apparent discrepancy between the severity of the translation and respiratory chain assembly defects, and suggest a function for EFG2 in quality control of translation elongation.