Figure 1
Manipulation of mTERF expression has minimal effects on steady-state levels of mature mitochondrial RNAs. (A) Schematic diagram of the promoter and rDNA region of human mtDNA. Because mTERF binding dictates the use of alternate transcriptional start sites and terminators, tRNAPhe and tRNALeu(UUR) fall into separate transcription units (PH1 and PH2 respectively). (B) Relative expression of mitochondrial transcripts in cells overexpressing mTERF, based on phosphorimaging of Northern blots probed successively for mitochondrial tRNAPhe and tRNALeu(UUR) and for 5S rRNA. Data (means ± SD) are signal ratios of tRNAPhe to tRNALeu(UUR) (F/L) and tRNAPhe to 5S rRNA (F/5S) for the mTERF-overexpressing clones shown in Additional File 1, Fig. S1, normalized to the corresponding ratio in cells stably transfected with empty-vector. Bars shown alongside are based on single reference experiments, using HEK293T cells transiently transfected with the same construct, or mock-transfected. (C) Q-RT-PCR analysis (means ± SD) of mitochondrial transcript levels, plus cytosolic18S rRNA, as indicated, in Flp-In™ T-Rex™-293 cells over-expressing mTERF-MycHis after doxycyclin induction for 3 d (or not induced). Data were normalized, in each case, to the corresponding ratio for uninduced cells. (D) Relative expression of mitochondrial transcripts in cells knocked down for mTERF, as indicated. – denotes mock-transfection. Northern blot probed successively for 16S rRNA and ND1 mRNA, as shown. The panels represent non-adjacent pairs of lanes from the same exposure of the same gel. (E) Relative expression of mitochondrial transcripts in cells knocked down for mTERF, as indicated, calculated from Northern blot data as in (B), normalized to the corresponding ratio in mock-transfected HEK293T cells. * indicates significant differences from the corresponding mock-transfected cells, and # a significant difference between cell-lines (t-test, p values as in text). For original blots see Additional File 1, Fig. S1C. Note that additional Q-RT-PCR data on levels of 12S rRNA gene transcripts are shown in Fig. 3.