FIG 5.
The eIF3cKD relaxes the stringency of the AUG start codon selection in vivo. (A) Schematic of the firefly luciferase reporter with alternative start sites AUG or GUG. (B) Schematic of the ATF4-firefly luciferase reporter showing positions of its two short uORFs, the first of which is mutated at the AUG start site and the other naturally extends into the coding region. (C) Quantification of the Sui− phenotype. HeLa cells were transfected with the indicated siRNAs, and 48 h later, the second round of transfection with individual reporters (AUG or GUG) together with a plasmid expressing the Renilla luciferase (used for normalization) was carried out. Luciferase activities were measured in WCEs 24 h after the second transfection. The mean percentages ± SD of the GUG initiation rates relative to those of AUG were calculated from three experiments. (D) Quantification of the leaky scanning phenotype. Same as described for panel C, except that the ATF4 reporter was transfected during the second round of transfection. The mean percentages ± SD of the increased or decreased skipping of the AUG start site of uORF2 in cells with knocked-down expression relative to control NT cells were calculated from three experiments.