The Deafness-Associated Mitochondrial DNA Mutation at Position 7445, Which Affects tRNA^Ser(UCN) Precursor Processing, Has Long-Range Effects on NADH Dehydrogenase Subunit ND6 Gene Expression

ABSTRACT

The pathogenetic mechanism of the deafness-associated mitochondrial DNA (mtDNA) T7445C mutation has been investigated in several lymphoblastoid cell lines from members of a New Zealand pedigree exhibiting the mutation in homoplasmic form and from control individuals. We show here that the mutation flanks the 3′ end of the tRNA^Ser(UCN) gene sequence and affects the rate but not the sites of processing of the tRNA precursor. This causes an average reduction of ∼70% in the tRNA^Ser(UCN) level and a decrease of ∼45% in protein synthesis rate in the cell lines analyzed. The data show a sharp threshold in the capacity of tRNA^Ser(UCN) to support the wild-type protein synthesis rate, which corresponds to ∼40% of the control level of this tRNA. Strikingly, a 7445 mutation-associated marked reduction has been observed in the level of the mRNA for the NADH dehydrogenase (complex I) ND6 subunit gene, which is located ∼7 kbp upstream and is cotranscribed with the tRNA^Ser(UCN) gene, with strong evidence pointing to a mechanistic link with the tRNA precursor processing defect. Such reduction significantly affects the rate of synthesis of the ND6 subunit and plays a determinant role in the deafness-associated respiratory phenotype of the mutant cell lines. In particular, it accounts for their specific, very significant decrease in glutamate- or malate-dependent O₂ consumption. Furthermore, several homoplasmic mtDNA mutations affecting subunits of NADH dehydrogenase may play a synergistic role in the establishment of the respiratory phenotype of the mutant cells.

ACKNOWLEDGMENTS

This study was supported in part by NIH grant GM11726 to G.A., grant 5RO1 DC 0142-04 from the National Institute on Deafness and Other Communication Disorders, NIH, to N.F.-G., and the New Zealand Deafness Research Foundation.

We thank Anne Chomyn for help in the statistical analysis, valuable advice, and useful discussions, and we thank Benneta Keeley, Arger Drew, and Rosario Zedan for technical assistance.