Fig. 3
The gene TSS variants quantitatively modulate the translational readout. The upper image depicts schematically the bicistronic reporter construct in which the gene 5′TL is fused to the firefly (FLuc) first cistron and second cistron renilla (RLuc) expression is assured by the EMCV IRES position inter-cistronically. Panels a-e: Bicistronic reporter assays performed in both MCF10A (blue bars) and MCF7 cells (red bars) to measure the impact of the TL variants on translation initiation events at the AUGGENE start. The upper image in each panel depicts the organisation of the various TL variants generated in the reporter. The positions (double headed arrow) and length (indicated in nucleotides within the bracket) of the uORFs are indicated. Red stars in panels C/D indicate uAUGs followed immediately by a stop codon and the arrow in panel D at the AUG14 indicates that this potential start codon is in the CAMKK1 ORF. The lower image in each panel graphically represents the normalised FLuc/RLuc ratios measured for each TL. Bars indicate the SEM for the biological triplicates. Experiments were repeated at least three times for each gene set. Only the CLDN7 V1/3 gave a statistically significant (P < 0.05) cell type variation in all three independent experiments (indicated as **). f An in-silico RNA fold of the CLDN7 V1/3 TL (RNAfold: http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi). The predicted Gibbs free energy is also indicated