Skip to main content
. Author manuscript; available in PMC: 2014 Jan 8.
Published in final edited form as: Nat Struct Mol Biol. 2011 Oct 7;18(11):10.1038/nsmb.2166. doi: 10.1038/nsmb.2166

Figure 4.

Figure 4

W-motifs are necessary for repression by full-length GW182 and function in bona fide miRNA repression. (a) W-motifs are required for repression by tethered full-length TNRC6C. The experiment was done as in Figure 2a but included the full-length TNRC6C. The right panel shows fold derepression relative to repression induced by WT NHA-TNRC6C or NHA-CED taken to be a value of 1 (broken line). Western analysis of expression levels of relevant mutants in a and other panels, with anti-HA antibody, is shown below the graphs. (b) Mutations in W-motifs lead to partial derepression of tethered mRNAs in D. melanogaster S2 cells. The assay was done as in Figure 2c but with the full-length dGW182 and TNRC6C. 6W, 7W and EF1388 mutations were described in Figures 2 and 3a but are here introduced into the full-length proteins. 13W mutant combines 6W and 7W; PAM2mut has EF960 WK967 Thr982 mutated. NHA-Q–rich (1080–1245) and NHA-CED represent TNRC6C fragments. In the right panel, data are presented as in a. (c) W-motifs are required to rescue depletion of endogenous dGW182. Endogenous dGW182 was depleted in D. melanogaster S2 cells with dsRNA (open bars); a batch of cells was treated with GFP-specific dsRNA as a control (black bars). Cells were transfected with RL-Con, FL-nerfin, and plasmids encoding miR-9b or miR-12, or the empty vector. To rescue depletion of dGW182, increasing amounts of plasmids encoding NHA-dGW182, NHA-TNRC6C or their mutants were co-transfected. In panels c and d, extracts from cells transfected with highest plasmid concentrations were used for western blotting. (d) W-motifs are necessary to complement the knockdown of endogenous TNRC6 proteins. HeLa cells were transfected with siRNAs targeting three endogenous TNRC6 proteins (open bars) or AllStars siRNA (negative control, black bars), RL-hmga2 reporter containing let-7 sites or its mutant version (RL-hmga2 mut), and increasing amounts of plasmids expressing NHA-TNRC6A or its mutants: 8W has Trp→Ala mutations in W-motifs within the CED region (Supplementary Fig. 1 and Online Methods); EF1358 has PAM2 mutated.