Figure 4. miR-965 regulates string and wingless.
(A, B) string (stg) and wingless (wg) transcript levels measured by quantitative real time RT-PCR in RNA isolated from w1118 control, KO1/KO2 and 965-rescue pupae at 21 hr APF. Data represent the average of three independent RNA collections ± SD. ANOVA: p < 0.01 comparing KO1/KO2 with control or with rescue for stg and wg. (C) Top: diagram of the predicted miR-965 target site in the wg 3′ UTR, showing pairing to the miRNA seed sequence. Residues shown in red were mutated in the mutant version of the wg 3′ UTR luciferase reporter. Below: luciferase activity in S2 cells transfected to express a tubulin-promoter miR-965 transgene, Renilla luciferase and the indicated firefly luciferase reporters. Control indicates the luciferase reporter with the SV40 3′ UTR, which lacks miRNA binding sites. wg UTR indicates the intact full-length wg 3′ UTR. Mut indicates the wg 3′ UTR with the miRNA seed site mutated as indicated in red. Data represent the average of 3 independent experiments ± SD. ANOVA: p < 0.001 comparing control to the intact 3′ UTR. p = 0.001 comparing the intact and site mutant versions of the 3′ UTR. (D) Top: diagram of the predicted miR-965 target site in the stg 3′ UTR, showing pairing to the miRNA seed. Residues shown in red were mutated in the seed mutant version of the reporter. The changes made in the extended target site mutant reporter are shown in Figure 3. Below: luciferase activity as in panel C. Data represent the average of 3 independent experiments ± SD. ANOVA: p < 0.0001 comparing control to the intact 3′ UTR and comparing intact to seed mutant and multiple mutant UTR reporters.
Figure 4—figure supplement 1. (A) Predicted miR-965 sites in the string 3′UTR.
