Fig. 1

Validation of miR-7, miR-34a, and miR-504 binding sites in the SHANK3 3′UTR by luciferase assays in HEK293T cells. a TargetScan prediction of the miRNA binding sites in the human SHANK3 3′UTR. The underlines indicate evolutionarily conserved binding sites. b miR-7 decreased the luciferase activity of the wild-type (hWT), but not the first binding site-mutant (hM), SHANK3 3′UTR. The luciferase activity of the rat Shank3 3′UTR (rWT), containing only the first binding site, was still reduced by miR-7. RL, Renilla luciferase; FL, firefly luciferase. c Mutation of the second miR-34a binding site (M2) blocked its repressive effect on the expression of the SHANK3 3′UTR. d Mutation of the second miR-504 binding site (M2) blocked its repressive effect on the expression of the SHANK3 3′UTR. e The validated functional miRNA binding sites in the SHANK3 3′UTR (left), and their base-pairings with the three miRNAs (middle). The sequence alignment across species for each miRNA binding site (right). f miR-34a did not affect the expression of the rat Shank3 3′UTR. g miR-7, miR-34a, and miR-504 synergistically decreased the expression of the SHANK3 3′UTR. h miR-7, miR-34a, and miR-504 did not affect the mRNA levels of the SHANK3 3′UTR. i miR-7 did not affect the expression of the human SHANK2 3′UTR that contains two putative miR-7 binding sites. All data are presented as mean ± SEM. Statistical analyses are in Additional file 1: Table S3