Experimental Validation of miR-7 Targets

(A) Schematic representation of the E(spl) and Brd gene complexes, which contain multiple predicted miR-7 target genes. bHLH-type transcriptional repressors are shown in red. Brd-type proteins are shown in blue. Other transcripts in the E(spl) cluster are in gray. Black asterisks indicate sites with no mismatch in the first eight residues (likely to be valid sites).

(B) miR-7 miRNA sequence showing the pattern of basepairing with target sites in E(spl) and Brd 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.

(C) Expression of the miR-7 sensor transgene is shown in green. Expression of the red fluorescent protein miR-7 miRNA under ptc–Gal4 control is shown in red. The right panel shows the miR-7 sensor alone.

(D and E) Expression of the m4 3′ UTR and hairy 3′ UTR sensor transgenes (green) were downregulated by miR-7 (red). Expression of the hairy 3′ UTR sensor was much lower than the m4 3′ UTR sensor overall. Cut protein, shown in blue, was downregulated in miR-7 expressing cells. The right panel shows a second example of Cut repression. The lower panel shows Cut channel alone.

(F) ClustalW alignment of miR-7 target sites in the 3′ UTRs of hairy 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.

(G) Cuticle preparations of a wild-type adult wing and a wing expressing miR-7 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.