The 5′ UTR of H-ferritin drives cap-independent translation of Firefly luciferase in a bicistronic construct. (A) Schematic diagram of the classic bicictronic construct. The first cistron encodes for Renilla luciferase, preceded by a cap structure and followed by three in-frame stop codons. The second cistron encodes for Firefly luciferase, followed by a poly(A) tail. An intracistronic sequence (ICS) is placed between the two cistrons (B). In vitro transcribed bicistronic RNAs with an ICS consisting of the 5′ UTR of SHMT1, or H-ferritin in sense (Ftn) or antisense orientation (aFtn) were transfected into HT1080 cells. Luciferase activity was monitored in cell lysates; values from three independent experiments (mean ± SD) are shown.
