Evidence that the 5′ UTR of H-ferritin harbors a functional IRES. (A) Schematic diagram of the bicistronic, positive feedback vector used to test for putative IRESs. The transcription of the bicistronic mRNA depends on a minimal promoter containing four copies of the Gal4 upstream activator sequence (4×UAS). The first cistron encodes for Renilla luciferase and the second for the transactivator Gal4/VP16. The 5′ UTR of H-ferritin (Ftn) and the inverted sequence of H-ferritin 5′ UTR (aFtn) were cloned in the intercistronic sequence (ICS). (B) Renilla luciferase activity following transfection of HT1080 cells with control (ICS), Ftn (5′UTR), or aFtn (5′UTR) constructs. The values from three independent experiments (mean ± SD) were normalized with Firefly luciferase activity from a cotransfected construct.
