Molecular dysfunction associated with the human mitochondrial 3302A>G mutation in the MTTL1 (mt-tRNA) gene

The gene encoding mt-tRNA, MT-TL1, is a hotspot for pathogenic mtDNA mutations. Amongst the first to be described was the 3302A.G transition which resulted in a substantial accumulation in patient muscle of RNA19, an unprocessed RNA intermediate including mt-16S rRNA, mt-tRNA and MTND1. We have now been able to further assess the molecular aetiology associated with 3302A.G in transmitochondrial cybrids. Increased steady-state levels of RNA19 was confirmed, although not to the levels previously reported in muscle. This data was consistent with an increase in RNA19 stability. The mutation resulted in decreased mt-tRNA levels, but its stability was unchanged, consistent with a defect in RNA19 processing responsible for low tRNA levels. A partial defect in aminoacylation was also identified, potentially caused by an alteration in tRNA structure. These deficiencies lead to a severe defect in respiration in the transmitochondrial cybrids, consistent with the profound mitochondrial disorder originally associated with this mutation. INTRODUCTION Mutations of mitochondrial DNA (mtDNA) are responsible for a broad range of diseases usually affecting multiple sites, particularly the central nervous system, muscle and retina. In some cases disease is limited to one tissue, even a single cell type (1). Although we continue to find new mutations in mtDNA, our understanding of how mitochondrial tRNA mutations cause their cellular defect has not grown to the same extent. In the present work, we further studied the 3302A>G mutation of the MT-TL1 gene that affects the 30 terminal part of the encoded mt-tRNA (2). This mutation is rare (2,3) and causes a severe myopathy with respiratory insufficiency. The rarity of this mutation and severity of its phenotype are in keeping with the fact that this nucleotide is highly conserved among mammals (4). Our previous studies showed that the 3302A>G mutation probably interferes with processing of an RNA precursor, RNA 19, that contains 16S rRNA, mt-tRNA and MTND1, and also showed that different precursor processing pathways are present in skeletal muscle and fibroblasts (2). Cytoplasts generated from fibroblasts of the patient were fused to 143B osteosarcoma rho cells and the cybrid clones were used in the present study to analyse in detail the molecular consequences of the mutation. *To whom correspondence should be addressed. Tel: +49 221 478 3610; Fax: +49 221 478 3538; Email: [email protected] *Correspondence may also be addressed to Prof. Laurence Bindoff. Tel: +47 559 75096; Fax: +47 559 75165; Email: [email protected]