title: Comparative analysis of the pathogenic mechanisms associated with the G8363A and A8296G mutations in the mitochondrial tRNA(Lys) gene.
abstract: Two mutations (G8363A and A8296G) in the mtDNA (mitochondrial DNA) tRNA(Lys) gene have been associated with severe mitochondrial diseases in a number of reports. Their functional significance, however, remains unknown. We have already shown that homoplasmic cybrids harbouring the A8296G mutation display normal oxidative phosphorylation, although the possibility of a subtle change in mitochondrial respiratory capacity remains an open issue. We have now investigated the pathogenic mechanism of another mutation in the tRNA(Lys) gene (G8363A) by repopulating an mtDNA-less human osteosarcoma cell line with mitochondria harbouring either this genetic variant alone or an unusual combination of the two mutations (A8296G+G8363A). Cybrids homoplasmic for the single G8363A or the A8296G+G8363A mutations have defective respiratory-chain enzyme activities and low oxygen consumption, indicating a severe impairment of the oxidative phosphorylation system. Generation of G8363A cybrids within a wild-type or the A8296G mtDNA genetic backgrounds resulted in an important alteration in the conformation of the tRNA(Lys), not affecting tRNA steady-state levels. Moreover, mutant cybrids have an important decrease in the proportion of amino-acylated tRNA(Lys) and, consequently, mitochondrial protein synthesis is greatly decreased. Our results demonstrate that the pathogenicity of the G8363A mutation is due to a change in the conformation of the tRNA that severely impairs aminoacylation in the absence of changes in tRNA stability. The only effect detected in the A8296G mutation is a moderate decrease in the aminoacylation capacity, which does not affect mitochondrial protein biosynthesis.
authors.affiliation: Departamento de Bioquímica, Instituto de Investigaciones Biomédicas Alberto Sols, CSIC-UAM, Facultad de Medicina, Universidad Autónoma de Madrid, 28029 Madrid, Spain.
authors.forename: Belén
authors.initials: B
authors.lastname: Bornstein
authors.forename: José Antonio
authors.initials: JA
authors.lastname: Mas
authors.forename: Clarice
authors.initials: C
authors.lastname: Patrono
authors.forename: Miguel Angel
authors.initials: MA
authors.lastname: Fernández-Moreno
authors.forename: Emiliano
authors.initials: E
authors.lastname: González-Vioque
authors.forename: Yolanda
authors.initials: Y
authors.lastname: Campos
authors.forename: Rosalba
authors.initials: R
authors.lastname: Carrozzo
authors.forename: Miguel Angel
authors.initials: MA
authors.lastname: Martín
authors.forename: Pilar
authors.initials: P
authors.lastname: del Hoyo
authors.forename: Filippo M
authors.initials: FM
authors.lastname: Santorelli
authors.forename: Joaquín
authors.initials: J
authors.lastname: Arenas
authors.forename: Rafael
authors.initials: R
authors.lastname: Garesse
mesh_terms: D049148:Aminoacylation; D045744:Cell Line, Tumor; D004272:DNA, Mitochondrial / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics*; D006801:Humans; D017243:MERRF Syndrome / Q000235:genetics / Q000503:physiopathology; D008928:Mitochondria / Q000378:metabolism*; D009154:Mutation; D010641:Phenotype; D011487:Protein Conformation; D012357:RNA, Transfer, Lys / Q000235:genetics* / Q000502:physiology
chemical_list: D004272:DNA, Mitochondrial; D012357:RNA, Transfer, Lys