FIG. 3.

The region between nucleotides −169 to −1 is required for efficient IRES function of the p27 mRNA. (A) Schematic diagram of the dicistronic mRNAs used for monitoring the IRES activities of truncated p27 mRNAs. Various regions of the p27 5′NTR were inserted into the intercistronic regions of dicistronic mRNAs composed of the Renilla (RLuc) and firefly (FLuc) luciferase genes. Black arrowheads represent the orientation of the RNA sequence. RNA VII contains the inverted p27 RNA sequence (nt −1 to −447) at the intercistronic region. (B) IRES activities of various regions of the p27 5′NTR. 293T cells were transfected with plasmids expressing the dicistronic mRNAs shown in panel A and the control plasmid pCMV•SPORT-βgal (Invitrogen). Forty-eight hours posttransfection, cells were harvested and firefly and Renilla luciferase activities were measured. Firefly and Renilla luciferase activities were normalized with β-galactosidase activity to adjust the transfection efficiency. Black and white bars depict firefly and Renilla luciferase activities, respectively. The relative ratios of firefly luciferase to Renilla luciferase (FLuc/RLuc) are also depicted by numbers on the top of each bar. The ratio of the luciferase activities (FLuc/RLuc) in cells containing the dicistronic mRNA (RNA VII in panel A) with an inverted p27 IRES (−1 to −447) is set to 1. The activities of firefly and Renilla luciferases are depicted in boxes below the graph.