Figure 7. Suppression of pORF translation by the uAUG in the L 5′-UTR is position dependent.

A. Diagram depicting wild-type and mutated L 5′-UTR-GFP reporter constructs in which the location of the uAUG was altered. For the L uORF, the black box represents the L uORF sequence, while the gray box depicts the remainder of the overlapping uORF that consists of GFP-derived sequences. Nucleotide changes that differ from the wildtype L 5′-UTR sequence are indicated in bold and the underlined sequences highlight the uORFs. Additional mutations were introduced to preserve the Kozak sequence at the −3 and +4 position such that they match that present in the wildtype L 5′-UTR. The reading frame of the uORF for each construct is indicated on the right. B. Equal amounts of in vitro transcribed mRNAs corresponding to the constructs depicted in panel A were transfected into 293T cells. At 2.5 hours post transfection, cells were harvested and analyzed by flow cytometry for GFP fluorescence. Each bar represents the mean of triplicate samples, and all values were calculated relative to the B-actin 5′-UTR GFP control mRNA which was set to 100%. C. A real time PCR measurement of GFP mRNA levels present in the transfected cells described in B was determined for each sample.