Role of GW182 domains in silencing. (A–F) S2 cells were treated with dsRNA targeting the 5′- and 3′-UTRs of GW182 mRNA. Control cells were treated with GFP dsRNA. These cells were subsequently transfected with a mixture of three plasmids: one expressing the indicated F-Luc reporters; another expressing miR-12 primary transcripts (+miR-12) or the corresponding empty vector (−); and a third expressing Renilla luciferase (R-Luc). Plasmids (25 ng) encoding wild-type HA-GW182, HA-GW182 mutants, or HA-MBP were included in the transfection mixtures, as indicated. Firefly luciferase activities were normalized to those of the Renilla luciferase transfection control and analyzed as described in Figure 2. (C,F) Northern blot analysis of representative RNA samples shown in B and E.
