Figure 4.
Impact of PTCH1b 5'UTR size and CGG-repeat number on cap-independent translation. Bicistronic pRuF-derived constructs were used to study the potential of the PTCH1b 5'UTR to drive cap-independent translation of the Firefly luciferase gene in transiently transfected MCF7 (A) or HEK 293T cells (B). Presented are the average ratios and standard deviations between Firefly (Fluc) and Renilla luciferase (Rluc) relative light units, normalized with the results obtained for empty pRuF vector, and standard deviation of at least 3 replicates. The PTCH1b 5'UTR size and CGG-repeat number are indicated. (C, D) The bicistronic nature of the transcript expressed by pRuF constructs was estimated measuring the Fluc/Rluc mRNA ratio by a qPCR approach. A ratio equal to 1.0 would strongly argue against the presence of a cryptic promoter within cloned PTCH1b 5'UTR resulting in a monocistronic Fluc mRNA transcript. A pRuF-type plasmid with cloned c-MYC 5'UTR was used as a positive control. For each UTR size type, results were compared to the corresponding reference allele with (CGG)7; *P < 0.05