Experimental Validation of <i>miR-7</i> Targets<b> </b>

<div><p>(A) Schematic representation of the <i>E(spl)</i> and <i>Brd</i> gene complexes, which contain multiple predicted <i>miR-7</i> target genes. bHLH-type transcriptional repressors are shown in red. Brd-type proteins are shown in blue. Other transcripts in the <i>E(spl)</i> cluster are in gray. Black asterisks indicate sites with no mismatch in the first eight residues (likely to be valid sites).</p> <p>(B) <i>miR-7</i> miRNA sequence showing the pattern of basepairing with target sites in <i>E(spl)</i> and <i>Brd</i> complex genes sorted in order of predicted folding energy. Yellow indicates a conventional basepair. Orange indicates a G:U basepair. Blue indicates a mismatch. The black bars indicate the position of loops in the target sites.</p> <p>(C) Expression of the <i>miR-7</i> sensor transgene is shown in green. Expression of the red fluorescent protein <i>miR-7</i> miRNA under ptc–Gal4 control is shown in red. The right panel shows the <i>miR-7</i> sensor alone.</p> <p>(D and E) Expression of the <i>m4</i> 3′ UTR and <i>hairy</i> 3′ UTR sensor transgenes (green) were downregulated by <i>miR-7</i> (red). Expression of the <i>hairy</i> 3′ UTR sensor was much lower than the <i>m4</i> 3′ UTR sensor overall. Cut protein, shown in blue, was downregulated in <i>miR-7</i> expressing cells. The right panel shows a second example of Cut repression. The lower panel shows Cut channel alone.</p> <p>(F) ClustalW alignment of <i>miR-7</i> target sites in the 3′ UTRs of <i>hairy</i> from several species. Asterisks indicate sequence identity. Black type indicates basepairs by Mfold (including G:U basepairs). Gray shading highlights the conserved miRNA–target binding region in all five species.</p> <p>(G) Cuticle preparations of a wild-type adult wing and a wing expressing <i>miR-7</i> under ptc–Gal4 control in the region between veins 3 and 4. Note the notching of the wing and the reduction of the region between veins 3 and 4, leading to partial fusion proximally. The size of the posterior compartment was increased apparently to compensate for reduction of the vein 3–4 region.</p></div>