Ex vivo and in vitro studies demonstrating IRES activity mediated by the FMR1 5' leader. (A) Capped dicistronic luciferase RNA containing the 5' leader from the β-globin or FMR1 mRNA inserted into the intercistronic region was transfected individually into C6 cells. Luciferase activity is shown as the ratio of Photinus luciferase to Renilla luciferase (P:R) and is normalized to the activity of the β-globin construct. The experiment was performed in triplicate, n = 3. (B) Monocistronic Photinus luciferase mRNA containing the β-globin or FMR1 5' leader was translated in rabbit reticulocyte lysate in the presence of increasing concentrations of cap analog. The initial level of Photinus luciferase activity from each monocistronic mRNA was normalized to 100. (C) Monocistronic Photinus luciferase constructs containing the β-globin, EMCV, or FMR1 5' leader were co-transfected with either a plasmid expressing hypophosphorylated 4E-BP1 or a control plasmid and assayed for luciferase activity. The activities obtained in cells co-transfected with 4E-BP1 is represented as a percentage of the activity obtained in cells co-transfected with the control plasmid. The experiment was performed in triplicate, n = 3.