Figure 3

RISC-associated DDX6 is necessary for pS387-dependent increase in binding of Limk1 mRNA to Ago2. (A) DDX6 knockdown abolishes S387 phosphorylation-dependent Limk1 mRNA binding to Ago2. Neurons transduced with lentiviral vectors expressing ^GFPAgo2(WT, S387A or S387D) and mCherry with or without DDX6 shRNA as shown were lysed and incubated with GFP-trap beads. RNA and proteins were isolated by trizol extraction, bound mRNAs were quantified by qPCR and proteins were detected by Western blotting using anti-GFP or anti-DDX6 antibodies. Representative Western blots are shown. Graph shows quantification of endogenous DDX6 bound to ^GFPAgo2. n = 5 independent experiments, *p < 0.05, **p < 0.01, two-way ANOVA followed by Tukey’s multiple comparison test. Data are mean ± S.E.M. (B) Graphs show quantification of Limk1 and Apt1 mRNA bound to ^GFPAgo2. Values are IP/input ratios, normalised to WT control condition. n = 5 independent experiments, *p < 0.05, **p < 0.01, ***p < 0.001, two-way ANOVA followed by Tukey’s multiple comparison test. Data are mean ± S.E.M. (C) Disrupting DDX6 association with RISC abolishes S387 phosphorylation-dependent DDX6 binding to Ago2 and Limk1 mRNA binding to Ago2. Neurons transduced with lentiviral vectors expressing ^GFPAgo2(WT, S387A or S387D) and mCherry, DDX6 shRNA or ^mCherryDDX6(WT or R386E) as shown were treated as in (A). Left panel shows representative blots, graph shows quantification of endogenous DDX6 (lane 1) or ^mCherryDDX6 binding to ^GFPAgo2. n = 5 independent experiments, **p < 0.01, ***p < 0.001, one-way ANOVA followed by Tukey’s multiple comparison test. Data are mean ± S.E.M. (D) Graph shows quantification of Limk1 mRNA bound to ^GFPAgo2. Values are IP/input ratios, normalised to WT control condition. n = 6 independent experiments, *p < 0.05, **p < 0.01, one-way ANOVA followed by Tukey’s multiple comparison test. Data are mean ± S.E.M.