Skip to main content
. 2013 Jul 15;140(14):2904–2916. doi: 10.1242/dev.092817

Fig. 1.

Fig. 1.

Identification of miR-310/13 in an RNAi-based targeted screen for miRNAs that suppress Wg pathway activity downstream of Axin. (A) The primary screen. miRNAs were tested for their ability to modulate Wg reporter (dTF12) activity in Clone 8 and S2R+ cells, where the pathway was ectopically activated by Axin dsRNA. (B) Unbiased cluster analysis of averaged log normalized scores for each screened miRNA in Clone 8 and S2R+ cells. (C) Highlighted clusters of strong inhibitors of the Wg reporter (top panel; orange box in B), including the previously reported Wg antagonist miR-8. Note the functional clustering of members of the miR-310/13 cluster. Also highlighted are potent activators of the Wg reporter (bottom panel; red box in B), including the previously published Wg agonist miR-315. (D,D′) Epistasis analyses. miR-310/13 strongly inhibits the dTF12-luciferase reporter when the Wg pathway is activated by dsRNA-mediated knockdown of Axin or by cDNA expression of ΔNLRP6 or Dsh. No significant inhibition by miR-310/13 was observed upon pathway activation with the constitutively active S37Aβ-cat. (E) Alignment of mature regions of Drosophila miR-310, miR-311, miR-312 and the human ortholog hsa-miR-25. d, Drosophila; a, Anopheles. (F,G) Alignments of multiple arm (F) and pan (G) 3′-UTR transcripts in various Drosophila and insect species revealing a conservation of miR-310/13 binding sites in arm and pan mRNA. (H) Predicted binding sites of miR-310/13 components in the 3′-UTR of arm and pan. (E-H) Gray shading highlights the conserved regions. (I-J′) pan 3′-UTR (I) and arm 3′-UTR (J) containing luciferase sensor reporters are significantly downregulated in the presence of miR-310/13, compared with control sensor containing full-length cDNA of arm lacking the 3′-UTR (Δ3′UTR, J′). (K) Western blot (WB) showing the ability of the miR-310/13 cluster and its individual components to significantly downregulate levels of Arm protein in S2R+ cells treated with Axin dsRNA. (L) hsa-miR-25 inhibits Wnt3a-induced activation of the SuperTOPFlash/STF16 reporter in human embryonic kidney (HEK293T) cells. Error bars indicate s.d. (n=4). P-values by Student’s t-test. RLU, relative luciferase units.