Figure 7.

Effect of an IRE on expression of bicistronic reporters carrying the p27 5′-UTR. Top: Diagram of potential mRNA products resulting from transcription of a bicistronic reporter in which an IRE has been inserted just downstream of the transcriptional start site within the TK promoter. The top diagram shows the expected transcript if there is no cryptic splice site within the 5′-UTR. In this case, only the upstream cistron (Renilla luciferase) would be affected by iron chelation. The bottom diagram shows the expected transcript if the 5′-UTR has a cryptic splice acceptor site. The IRE would be linked to a monocistronic mRNA encoding only firefly luciferase and iron chelation would inhibit expression of this enzyme. Bottom: NIH3T3 cells were transfected with bicistronic reporters pTKLL-472 and pTKLL-472-IRE. These constructs are identical except that pTKLL-472-IRE has an IRE inserted 35 nt downstream of the TK transcriptional start site. The transfected cells were treated with (+, gray bars) or without (−, black bars) the iron chelator DFO. The cells were harvested and Renilla (R) and firefly (F) luciferase activities were determined. For each transfection, the value of luciferase activity in the absence of DFO was set to 1.