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 F-Luc-Nerfin-1 reporter; another expressing miRNA primary transcripts (+miR-9b or miR-279) 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 set to 100 in cells transfected with the empty vector (i.e., in the absence of the miRNAs). (A,D) Normalized Firefly luciferase activities in the absence or presence of miRNAs in control cells (i.e., cells treated with GFP dsRNA and transfected with MBP). (B,E) Relative fold de-repression for each condition. Mean values ± standard deviations from three independent experiments are shown. (C,F) Northern blot analysis of representative RNA samples shown in B and E.
