FIGURE 5.

miR-2-mediated regulation from single or dual binding sites in vitro and in vivo. (A) The GL-FL-reporter mRNAs containing one, two, or six miR-2-binding sites were incubated in the Drosophila cell-free extract. A single binding site is only effective from the 3′UTR. Two binding sites yield over twofold repression from the 3′UTR and mediate ∼1.5-fold repression from the 3′ end of the ORF, but are ineffective from the 5′UTR and the 5′ end of the ORF. (B) The GL-FL-reporter mRNAs containing one, two, or six miR-2-binding sites were transfected into Drosophila S2 cells. A single binding site triggers modest repression from the 3′UTR and the 5′ end of the ORF. Two binding sites trigger robust repression from the 3′UTR, but are only moderately effective from the 5′UTR and the 5′ end of the ORF. Shown are averages and standard deviations of five independent experiments.