Fig. 6.

Exon B sequential sequence deletion analysis: effect on transcription and luciferase activity. A: experimental deletion-mutant (del_mut) constructs (n = 8), generated from the rpcDNA3/5′-UTR/LUC (AeBD) by site-directed mutagenesis. Del_2 to del_7 refer to 5-nucleotide consecutive deletions within eB. Del_1 corresponds to the removal of the last 5 nucleotides of exon A, whereas del_8 refers to deletion of the first 5 nucleotides of exon D. B: luciferase mRNA levels, expressed as the ratio of firefly to Renilla relative to LUC (no 5′UTR control), after 30 h of transfection of NCI-H441 with del_mut constructs. Del_1, del_3, del_7, and del_8 constructs exhibited lower transcription of luciferase than the natural AeBD 5′UTR (positive control) (*P < 0.05), but significantly higher transcription than the negative control construct (LUC) (#P < 0.01). C: luciferase activity (expressed as the firefly to Renilla ratio) after 36 h of transfection with control or del_mut constructs shown in A. All the del_mut constructs tested exhibited lower luciferase levels compared with the positive control AeBD (*P < 0.05; **P < 0.001). Luciferase activity levels were barely detectable for the construct without 5′UTR (LUC, #P < 0.001), relative to each of the other constructs. Luciferase activity was measured in triplicate for each sample. Each bar shows mean + SE (n = 3 independent experiments).