Figure 7

Translational repression by uORFs of selected T1 transcripts. (A) Identification of uAUG (depicted by diamonds) and uORFs (boxes, with the vertically positioned triangles indicating AUG) in the 5'-UTR sequences (horizontal lines) of selected T1 transcripts. Dotted boxes are uORFs found in the testicular T1 transcript. The depicted 5'-UTR sequences were placed before the luciferase (Luc) gene in the pGL3-Control (SV40Pr-Enh) or pGL3-Promoter (SV40Pr) vectors. In the T1E16-RT4 construct, a new initiation codon was fortuitously created by the cloning process adding 16 amino acid residues (dashed box) to the luciferase protein which did not affect the luciferase activity. (B) Transient transfection of CHO-K1 cells and luciferase assays using the constructs described in (A). Transfection with the pGL3-Control or pGL2-Promoter plasmid was used as a positive control (Cont.). The data shown are from three independent experiments. (C) RT-PCR analysis of relative luciferase mRNA levels in the transfected CHO-K1 cells. Transfected cells were harvested at the indicated time points for total RNA preparation and semi-quantitative RT-PCR analysis using luciferase gene-specific primers (Table 2). (D) Western blot analysis of the luciferase protein levels in the transfected CHO-K1 cells at different post-transfection time points using an anti-luciferase antibody. The control (Cont.) data were derived from mock transfection using the blank vector. (E) Effects of disruption of uORFs on expression of the luciferase reporter gene. The three uAUGs and the two uORFs of the T1 transcript, shown in (A) above, were disrupted by site-specific mutagenesis at the initiation codons as shown: mut a was a double mutant of the two uAUGs (underlined) found in the first uORF; mut b removed the uAUG in the downstream uORF whereas mut ab was a mutant in both the up- and downstream uORFs. The mutagenised constructs were used in transient transfection of the CHO-K1 cells followed by luciferase assays. The data shown in the bottom panel were derived from three independent transfection experiments. The parental T1 and the uORF-free T1E16-RT4 were included for comparison.