Figure 2.

GW182 enhances let-7 miRNA-mediated translational repression. (A) Schematic representations of the firefly luciferase mRNAs FLuc-6xT and FLuc-6xTmut6. (B) Translation of capped and polyadenylated FLuc-6xT mRNA in the absence (−) or presence (+) of let-7. Cell extracts prepared from control 293F cells and 293F cells overexpressing Flag-Ago2 and Flag-GW182 (see Fig. 1A) were used in the combinations indicated above the figure. The FLuc and RLuc activities were measured, and the FLuc-to-RLuc activity ratio in the reaction without let-7 was set at 100. The data shown constitute an average of at least three independent experiments, with standard deviations. (C) Biotinylated FLuc-6xT mRNAs (capped and polyadenylated) were incubated with the mixed extract (Ago2 + GW182; see lane 1) in the absence (−) or presence (+) of let-7. The mRNAs were captured with SA-PMPs. (Left panel) The proteins bound to the mRNAs were detected using an anti-Flag M2 antibody (lane 1, Ago2 + GW182 extract; lanes 2–4, pull-down). The sizes (in kilodaltons) of the molecular markers (M) are indicated on the left. (Right panel) The mRNAs captured with SA-PMPs were detected by dot hybridization (see the text).