Figure 4: Using ASRE to silence a mitochondrial gene.

(a) A diagram of mitochondrial genome. The circular DNA genome of human mitochondrion contains genes coding for 13 proteins (colour coded for different oxidative phosphorylation complexes), two rRNA (16S and 12S) and 22 tRNAs. A unique sequence (TTTATGTG) in mtND5 coding region was chosen as the ASRE target. (b) Diagram of mitoASREs containing an N-terminal mitochondrial targeting peptide that, after translocation, will be cleaved (shown by an arrow). (c) Immunofluorescence of 293 cells expressing mitoASREs and a control ASRE. The mitochondria were stained with MitoTracker. (d) The changes of mtND5 RNA level 24 h after the tetracycline induction. Two independent inductions were carried out, and the real-time RT–PCR experiments were conducted in triplicates with glyceraldehyde 3-phosphate dehydrogenase as internal controls. The average of the relative mtND5 RNA levels compared with uninduced samples were plotted, with error bars indicating standard derivations. (e) Changes of mtND5 protein after induction. The levels of both mitochondrial encoded proteins (COX1 and cytB) and the nuclear encoded protein (tubulin) were measured as controls. (f) Changes of the complex I activity upon induction as measured with a spectrophotometric assay²⁵. For each cell line, the activities relative to uninduced sample were plotted. Three independent inductions and mitochondrial preparations were conducted, and each sample was measured in triplicates. The mean and s.e. of all samples were plotted (*P=0.0014, paired Student's t-test).