Selective Pharmacological Targeting of a DEAD Box RNA Helicase
Figure 4
Functional requirements for eIF4A activity in translation.
(A) Schematic representation of the reporter construct used in these studies is shown on top. (B) Left Panel: Rescue of hippuristanol-induced translation inhibition by eIF4AIIG/T. In vitro translations in RRL programmed with capped FF/HCV/Ren mRNA (8 µg/ml) and containing vehicle (0.1% DMSO) or 5 µM hippuristanol (in 0.1% DMSO) were supplemented with 0.5 µg recombinant protein. Protein synthesis was assessed by using 35S-methionine incorporation as well as by monitoring luciferase assays. Protein products were separated by SDS-PAGE and visualized by autoradiography. The arrow indicates the position of migration of the firefly luciferase, whereas the arrowhead denotes the position of migration of Renilla luciferase. Right panel: Relative luciferase activity obtained in the presence of recombinant eIF4A. Firefly RLU readings obtained in the presence of recombinant eIF4A and hippuristanol were standardized to Renilla RLU values and set relative to the values obtained in the presence of vehicle (DMSO). The average of 3–8 experiments is shown with the standard deviations denoted. (C) eIF4AI and eIF4AII are functionally interchangeable. In vitro translations in RRL containing vehicle (DMSO) or 5 µM hippuristanol were supplemented with 0.8 µg recombinant eIF4A where indicated, and programmed with capped FF/HCV/Ren mRNA (8 µg/ml). Left panel: Protein products were separated by SDS-PAGE and visualized by autoradiography. The arrow indicates the position of migration of the firefly luciferase, whereas the arrowhead denotes the position of migration of Renilla luciferase. Right panel: Relative luciferase activity obtained in the presence of recombinant eIF4A and hippuristanol was standardized to Renilla Luciferase levels and set relative to the values obtained in the presence of vehicle (DMSO). The average of 4–5 experiments is shown with the standard deviations denoted. (D) Translational rescue by eIF4AIIG/T is selective for hippuristanol. In vitro translations in RRL containing vehicle (DMSO), 5 µM hippuristanaol, or 0.4 µM pateamine were supplemented with 0.8 µg recombinant eIF4A where indicated, and programmed with capped FF/HCV/Ren mRNA (8 µg/ml). Protein products were separated by SDS-PAGE and visualized by autoradiography. The arrow indicates the position of migration of the firefly luciferase, whereas the arrowhead denotes the position of migration of Renilla luciferase. The figure is a representative display of one of two experiments.