FIGURE 4.

miR-2 represses translation from 5′UTR- and ORF-binding sites in vivo. (A) In vitro transcribed GL-FL-reporter mRNAs are repressed by endogenous miR-2 following transfection into Drosophila S2 cells. Luciferase activity of the ORF(3′) constructs is not significantly over background, possibly due to misfolding and degradation of luciferase C-terminal fusion proteins in S2 cells. (B) The repression is mediated by miR-2, since it is specifically relieved by cotransfection of 100 nM anti-miR-2 LNAs. (C) miR-2 represses luciferase expression from transfected reporter plasmids. (D) This repression is also specifically relieved upon cotransfection of 100 nM anti-miR-2 LNAs. (E) Total RNA was extracted from S2 cells transfected with the reporter plasmids and subjected to RT–qPCR analyses. The wt reporter mRNAs are equally stable throughout the experiment. mRNA levels could also be measured for the ORF(3′) constructs, as expected. Shown are averages and standard deviations from five independent experiments. Statistical significance was evaluated through an unpaired two-tailed t-test: (*) P < 0.05.