Abstract
Characterization of the basal transcription machinery of mitochondrial DNA (mtDNA) is critical to understand mitochondrial pathophysiology. In mammalian in vitro systems, mtDNA transcription requires mtRNA polymerase, transcription factor A (TFAM), and either transcription factor B1 (TFB1M) or B2 (TFB2M). We have silenced the expression of TFB2M by RNA interference in Drosophila melanogaster. RNA interference knockdown of TF2BM causes lethality by arrest of larval development. Molecular analysis demonstrates that TF2BM is essential for mtDNA transcription during Drosophila development and is not redundant with TFB1M. The impairment of mtDNA transcription causes a dramatic decrease in oxidative phosphorylation and mitochondrial ATP synthesis in the long-lived larvae, and a metabolic shift to glycolysis, which partially restores ATP levels and elicits a compensatory response at the nuclear level that increases mitochondrial mass. At the cellular level, the mitochondrial dysfunction induced by TFB2M knockdown causes a severe reduction in cell proliferation without affecting cell growth, and increases the level of apoptosis. In contrast, cell differentiation and morphogenesis are largely unaffected. Our data demonstrate the essential role of TFB2M in mtDNA transcription in a multicellular organism, and reveal the complex cellular, biochemical, and molecular responses induced by impairment of oxidative phosphorylation during Drosophila development.
Highlights
The majority of cellular ATP is generated by oxidative phosphorylation (OXPHOS)4 in the inner mitochondrial mem
TFAM is a member of the high mobility group family of proteins and is inverted repeat; TFB1/2M, mitochondrial transcription factor B1/2; TFAM, mitochondrial transcription factor A; UAS, upstream activating sequences; RNAi, RNA interference; qPCR, quantitative PCR; d, Drosophila; PBS, phosphate-buffered saline; RT, reverse transcriptase; ANOVA, analysis of variance; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine
The establishment of an in vitro transcription system employing recombinant proteins showed that both factors can stimulate specific transcription from light and heavy strand promoters, in a reaction that is strictly dependent on TFAM, and arguing that the basal transcription machinery in mammals is composed of mtRNA polymerase, TFAM, and either TFB1M or TFB2M, with the latter being at least 1 order of magnitude more active in the in vitro system [15, 16]
Summary
Fly Stocks and Culture—Flies were raised on standard yeastglucose-agar medium at 25 °C and 65% relative humidity in 12-h light/dark cycles unless otherwise indicated. The fixed preparations were blocked in PBS, 5% goat serum, 2% bovine serum albumin, 0.4% Triton X-100 for 30 min, incubated overnight at 4 °C with rabbit anti-horseradish peroxidase (1:200; Jackson ImmunoResearch), washed four times in PBS, 0.4% Triton X-100 for 1 h, and incubated with the secondary antibody goat anti-rabbit IgG coupled to Alexa Fluor 568 (1:400; Molecular Probes) for 2 h at room temperature in the dark. Immunocytochemistry in Imaginal Wing Discs—Imaginal discs from third instar larvae were dissected in PBS and fixed with 4% paraformaldehyde in PBS for 20 min at room temperature They were blocked in PBS, 1% bovine serum albumin, 0.3% Triton X-100 for 1 h, incubated with the primary antibody overnight at 4 °C (dilution 1:50), washed four times in blocking buffer and incubated with the appropriate secondary antibody for 2 h at room temperature in the dark (dilution 1:200). The one-way ANOVA test and the Bonferroni post-test were used to determine statistical significance of the results
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