Let-7-mediated translational repression is inhibited by a GW182 fragment containing an Ago2-binding site. (A) FLAG/SBP-Ago2 was coexpressed with FLAG-GW182 (1–910) or FLAG-GW182 (1–910, W470A, W755A, W828A) in HEK293F cells. SBP pull-down assays were performed, as described in the Figure 7A legend, and the precipitates were analyzed by Western blotting using an anti-FLAG antibody. Lane L, cell lysate; lane E, fraction eluted with biotin. (B) In vitro translation experiments were performed with HEK293F extracts (containing 10% [v/v] Ago2 extract) along with 2%, 4%, and 8% (v/v) of the wild-type, or 4%, 8%, and 16% (v/v) of the mutant FLAG-GW182 (1–910) extract. Capped FLuc-6xT with a poly(A) tail mRNA was translated in the absence (−) or presence (+) of let-7. 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 three independent experiments, with standard deviations. (C) Comparison of the translation efficiencies of the extracts with different ratios of FLAG-GW182 (1–910) extract in the absence of let-7. The average number of light units produced by the translation of FLuc mRNAs in the absence of FLAG-GW182 (1–910) lysate was ∼2,200,000. (D) Expression of FLAG-Ago2 and wild-type or mutant FLAG-GW182 (1–910) in the cell extract mixture using the translation experiment. The expressed proteins were detected by Western blotting using an anti-FLAG M2 antibody. (E) Analysis of capped and polyadenylated FLuc-6xT and RLuc mRNAs by Northern blotting with an FLuc probe and an RLuc probe. Translation reactions of mRNAs were performed with the cell extract mixture containing 8% (v/v) of the wild-type GW182 (1–910) and 16% (v/v) of the mutant GW182 (1–910). As molecular markers, nonadenylated (shorter) and polyadenylated (longer) transcripts were loaded in lane M. After 60 min (upper panel) and 120 min (lower panel) of translation, the RNAs were extracted from the reaction mixture, and similar amounts were loaded in each lane.
