Abstract
We report the cloning and molecular analysis of Drosophila mitochondrial DNA helicase (d-mtDNA helicase) homologous to human TWINKLE, which encodes one of the genes responsible for autosomal dominant progressive external ophthalmoplegia. An RNA interference construct was designed that reduces expression of d-mtDNA helicase to an undetectable level in Schneider cells. RNA interference knockdown of d-mtDNA helicase decreases the copy number of mitochondrial DNA (mtDNA) approximately 5-fold. In a corollary manner, overexpression of d-mtDNA helicase increases mtDNA levels 1.4-fold. Overexpression of helicase active site mutants K388A and D483A results in a severe depletion of mtDNA and a dominant negative lethal phenotype. Overexpression of mutants analogous to human autosomal dominant progressive external ophthalmoplegia mutations shows differential effects. Overexpression of I334T and A442P mutants yields a dominant negative effect as for the active site mutants. In contrast, overexpression of A326T, R341Q, and W441C mutants results in increased mtDNA copy number, as observed with wild-type overexpression. Our dominant negative analysis of d-mtDNA helicase in cultured cells provides a tractable model for understanding human autosomal dominant progressive external ophthalmoplegia mutations.
Highlights
Autosomal dominant progressive external ophthalmoplegia is a human mitochondrial disorder associated with the presence of multiple deletions of mitochondrial DNA (mtDNA) [2,3,4,5]
To compare the effects of the human Autosomal dominant progressive external ophthalmoplegia (adPEO) mutations with those we observed with active site mutants in Schneider cells, we constructed metallothionein-inducible plasmids expressing five d-mtDNA helicase variants carrying A326T, I334T, R341Q, W441C, and A442P substitutions, which are analogous to the A359T, I367T, R374Q, W474C, and A475P mutations found in adPEO patients, respectively, and map within the linker region and helicase domain (Fig. 2)
In Drosophila Schneider cells treated with RNAi, the expression of d-mtDNA helicase is suppressed to an undetectable level, inducing cellular phenotypes of slow growth, reduced viability, and a 5-fold reduction in mtDNA copy number
Summary
Identification and Sequence Analysis of d-mtDNA Helicase cDNA—The amino acid sequence of TWINKLE was used to search the Berkeley Drosophila Genome Project Data base. One sequence (CG5924) was identified that has a high level of homology to TWINKLE [7]. Full-length cDNA was prepared using the SMART RACE cDNA amplification kit (Clontech), total RNA from Drosophila Schneider S2 cells, and the following primers: 5Ј-TGACTGGCATCGTAGTGCAAC-3Ј and 5ЈgggctcgagGCTCTTTGAAGCGTTCCAAGG-3Ј for 5Ј-rapid amplification of cDNA ends (RACE) and 5Ј-ACGCATTGCGTTGTATGCCTGC-3Ј and 5Ј-gggtctgagTACGAACTGAAG-.
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