The effect of a strong hairpin near the 5′ cap on the expression of the second cistron in CIRP 5′-UTR containing bicistronic constructs at 37 (gray) and 32°C (black). (A) Determination of first cistron renilla luciferase expression in the pRF control plasmid and hairpin containing pHrpRMF, pHrpRF-L, and pHrpRF-S showed that the hairpin dramatically reduces expression levels of the first cistron. (B) Second cistron firefly expression was unhindered by the presence of the hairpin from the c-myc containing control vector (pHrpRMF) and the CIRP long 5′-UTR containing vector (pHrpRF-L), resulting in expression levels significantly enhanced above those in the absence of the IRES. On the other hand, the hairpin prevented firefly expression from the CIRP short 5′-UTR (pHrpRF-S, B). All data shown are the means of triplicate experiments ± SD.
