Experimental Validation of <i>miR-2</i> Targets

<div><p>(A) Conservation of sequences in the 3′ UTRs of <i>reaper</i>, <i>grim</i>, and <i>sickle</i> genes of D. melanogaster and D. pseudoobscura. Blocks of high sequence similarity are color-coded. The location of predicted <i>miR-2/miR-13</i> family target sites are underlined in black.</p> <p>(B) ClustalW alignment of predicted <i>miR-2/miR-13</i> family target sites in the <i>reaper</i>, <i>grim</i>, and <i>sickle</i> 3′ UTRs. <i>Z</i> scores for <i>miR-2a</i> and <i>miR-2b</i> are shown for each site. The first bases of the <i>grim</i> and <i>sickle</i> sites do not pair with the miRNAs. Because we use a hairpin-forming linker sequence, this causes a penalty in Mfold, which gives these sites lower <i>Z</i> scores than they should otherwise have.</p> <p>(C) Immunoblot of S2 cells transfected to express a tubulin promoter–EGFP–reaper 3′ UTR construct (lane 2) or a comparable construct from which the <i>miR-2/miR-13</i>-binding site in the UTR was deleted (lane 3). Control cells were transfected with empty vector (lane 1). The blot was probed first with antibody to GFP and then reprobed with anti-tubulin as a loading control.</p> <p>(D and E) Expression of the <i>grim</i> and <i>sickle</i> 3′ UTR sensor transgenes (green) was downregulated by <i>miR-2b</i> expressed under ptc–Gal4 control (red).</p>