Validation of direct targets of miR310s by in vitro mutagenesis. (A) Putative target sites of miR310 on the 3′ UTRs of Dl, E2f2, EcR, Mad, and Mef2. miR310 seed region, target sites, and mutant sites were shaded in red. The mutant site of each gene contained altered base pairs, which disrupt pairing with miR310s. The 3′ UTR of Mef2 has two target sites, both of which were mutated. (B) The expressions of the five target genes are de-repressed in mir310s- flies (in ovaries of 3-d-old females) and repressed again by either Dm310s or Dp310s. (Relative expression were measured by qRT-PCR in triplicate, using rp49 as endogenous control.) (C) The luciferase reporter with wild-type miR310s target sites is significantly inhibited by the cotransfected mir310s vis-à-vis those carrying mutated target sites. Five replicates in Drosophila S2 cells. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001 by Student's t-test.
