RNase protection assay on mitochondrial (−)strand transcripts mapping around the cyt b/ND1-binding site in DmTTF-depleted cells. (A) Schematic representation of digestion products using probes Ribo-1 (295 nt) and Ribo-2 (218 nt). Riboprobes (grey bold arrows), mature transcripts (continuous arrows) and read-through transcripts (dotted arrows) are indicated above the cyt b/ND1 region map. Dashed regions indicate non-coding sequences. (B) Total cellular RNA (50 µg), extracted from untreated (control) and DmTTF-dsRNA treated (RNAi) D.Mel-2 cells, was hybridized with about 1.5 × 10⁵ c.p.m. of Ribo-1 and Ribo-2 probes and digested with RNase A and T1. Digestion products were denatured and run on a 10% polyacrylamide/7 M urea gel. Y, sample containing 50 µg of yeast total RNA. M, Decade RNA marker (Ambion).

Effect of DmTTF depletion on the level of mitochondrial sense and antisense transcripts, and on mtDNA copy number. (A) Relative quantification of mitochondrial RNAs by real-time RT–PCR. MtDNA molecule is represented as a linear map. Transcription direction of (−) and (+)strand is indicated by black arrows; the two DmTTF-binding sites are marked by vertical dotted lines. Black bars reported above (−)strand and below (+)strand, in correspondence of the analyzed genes, indicate the relative content (R) of sense and antisense transcripts, normalized to 28S rRNA (endogenous control), in DmTTF depleted (RNAi) with respect to control cells, fixed as value 1. The relative quantification was performed according to the Pfaffl equation R = (E_T)^ΔCt,T/(E_C)^ΔCt,C (24), where E_T is the amplification efficiency of target gene transcripts; E_C is the amplification efficiency of endogenous control; ΔC_t,T and ΔC_t,C are the differences between C_t of the control and C_t of the RNAi sample for target gene transcripts and for endogenous control, respectively. For each amplicon, the mean ratio value was obtained from at least four real-time RT–PCR assays, using RNA obtained from independent RNAi experiments; standard deviations are indicated on the top of the bars. Statistical analysis showed that the differences between RNAi and control are all significant (P < 0.05) except for ATPase6/8 and COIII-antisense transcript. (B) Southern blotting analysis of mtDNA. Total DNA (5 µg) from untreated (control) and treated (RNAi) D.Mel-2 cells was digested with XhoI, fractionated on a 0.7% agarose gel and electroblotted to nylon membrane. The filter was hybridized with radiolabeled probes for both mitochondrial ND3 gene and nuclear H2B histone gene (endogenous control). The amount of c.p.m. used for each probe was such to obtain a comparable signal for mitochondrial and nuclear DNA.

Effect of DmTTF-targeted RNAi in D.Mel-2 cells. (A) Western blotting analysis of D.Mel-2 total cell and mitochondrial lysate. A total of 250 µg of proteins were fractionated on a 12% SDS–polyacrylamide gel, electroblotted to PVDF filters and incubated with polyclonal antibodies against recombinant DmTTF. (B) D.Mel-2 cells were either untreated (control) or treated with odds-paired (Opa1) or DmTTF dsRNA. Mitochondrial lysate (250 µg of proteins) was probed with polyclonal antibodies against DmTTF, h-NDUFS4 or D-TFAM.

RT–PCR analysis on mitochondrial transcripts spanning the cyt b/ND1-binding site in DmTTF-depleted cells. (A) Schematic illustration of the cyt b/ND1-binding site. Black arrows represent the forward (11 494–11 520 nt) and reverse (11 855–11 834 nt) primers used for RT–PCR. Dashed regions indicate non-coding nucleotides. (B) Total RNA (600 ng) extracted from untreated (control) and treated (RNAi) D.Mel-2 cells was used as template in RT–PCR; 10 µl-samples were collected at the indicated cycles, run on a 1.5% agarose gel and stained with ethidium bromide. Nuclear encoded 28S rRNA was used as endogenous control.

Proposed models of mtDNA transcription mechanism in Drosophila. Grey areas show the coding sequences on either strands; arrows indicate directions of (+) and (−)strand transcription initiating from the respective promoters. Dotted lines indicate attenuated transcription. T, termination; A, attenuation. See text for explanation.