FIGURE 1.

miR-2 represses translation from 5′UTR- and ORF-binding sites in vitro. (A) Schematic representation of the reporters used in this study. The firefly luciferase open reading frame is indicated by a white box (GL-FL); six binding sites for miR-2 are shown as black bars. (B) miR-2 represses translation from both UTRs and from ORF-binding sites in the Drosophila cell-free extracts. (C) Kinetic analysis of translational repression. Samples are analyzed at 10-min intervals during the repression assay. One-hundred percent repression for each construct corresponds to the values indicated in B. (D) The repression observed requires miR-2, since it is specifically relieved by anti miR-2 LNAs. (E) Luciferase activity from the different mut constructs is indicated to show the effect of the position of the miR-2-binding sites on enzymatic activity. (F) The RT–qPCR analyses show no significant destabilization of the reporter mRNAs at the end (t2) of the repression assay. Shown are averages and standard deviations of five (B,D) or three (C,F) independent experiments. (E) One experiment performed in duplicate from B.