FIGURE 3.

Effective repression of all of the reporters requires the interaction between AGO1 and GW182 and targets cap-dependent translation. (A) In vitro translation reactions were incubated with either a dominant-negative version of the Drosophila GW182 protein [GW182(1-592)] or with protein buffer. The addition of 200 nM dominant-negative protein significantly derepresses all wt reporters, while no effect is observed with buffer alone. The graph shows averages and standard deviations from three independent experiments. (B) GL-FL-reporter mRNAs bearing either the m⁷G-ARCA cap or the 21-ARCA cap analog (Zdanowicz et al. 2009) were synthesized in vitro and incubated in the Drosophila embryo extract. The luciferase counts of the mut constructs (dark-gray bars) and the fold repression of each wt-mut pair (light-gray bars) bearing cap 21-ARCA were compared with their m⁷G-ARCA-capped counterparts. In all cases, cap 21-ARCA displays no effect on general translation, while augmenting miR-mediated repression. The graph shows averages and standard deviations from six independent experiments. Statistical significance was evaluated through an unpaired two-tailed t-test: (*) P < 0.05.