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. 2009 Mar 1;23(5):619–632. doi: 10.1101/gad.1760209

Figure 2.

Figure 2.

Muscle miRNA target validation. (A) Luciferase reporter assays to validate individual miR-1 and miR-133 targets. The reporter mRNA contains the Firefly luciferase (Fluc) ORF and the target 3′UTR sequence. Each reporter was coinjected with Renilla luciferase and buffer (gray bar) or a miRNA duplex (miR-1, red; miR-133, blue; both, violet). Fluc activity was normalized to Renilla luciferase and Fluc activity without miRNA duplex is set to 100%. Light-colored bar indicates repression by miRNA (P < 0.05, n = 3). Error bar shows SD. Gene name and number of target sites are shown below each bar; asterisks indicate targets expressed twofold lower in muscle versus nonmuscle. Dark gray bars (right), represent the level of repression when the seed sequences in the 3′UTR were mutated from CATTCC to CTATCC. Green bars show a positive control with three targets partially complementary to miR-1 or miR-133 (light green, IPTx3) and a negative control with no miRNA target sites (dark green, no site). (B) Dual miRNA reporter construct used to analyze the target regulation by endogenous miRNAs. A plasmid DNA containing GFP (control, green) and RFP-3′UTR (reporter, red) expressed from two SV40 promoters was injected into wild-type or miRNA-MO-injected embryos at the one-cell stage. Because injected DNA is inherited in a mosaic pattern, the dual reporter ensures that each cell that expressed GFP also expresses RFP. GFP and RFP expression in muscle was analyzed at 48 h post-fertilization (hpf) . (C–E) Control GFP expression (green) and reporter RFP expression (red) at 48 hpf. The panels show an enlarged view of somites. RFP expression is repressed in wild-type muscle cells (left) while the repression is abolished in the presence of miRNA-MO (right) (dashed boundaries). Arrowheads indicate muscle cells, which are elongated. Asterisks indicate nonmuscle cells. In all cases tested (n = 3), repressing the endogenous miRNA upon injection of the cognate miRNA-MO (+miR-1-MO or +miR-133-MO) abolished miRNA-mediated repression of the RFP reporter in muscle.

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