Figure 4

Members of the PTEN/AKT signaling pathway and splicing factors are direct downstream targets of miR-486 in skeletal muscle. (A) Schematic shows the location of the 3'UTR miR-486 target cloned downstream of the luciferase gene reporter with a poly(A) tail. (B) Luciferase miRNA reporter assay of several of the 3'UTR of the predicted miR-486 direct downstream targets. The lower graph shows results from the mutation of the miR-486 seed site, which ablates miR-486 binding and functions to derepress luciferase expression. The y-axis displays relative RLU activity normalized to 100% activation of the reporter transfected alone (vector alone). Error bars indicate SEM (n = 3 replicates, with the experiment performed on three separate occasions using HEK293T cells). *P < 0.005 and **P < 0.05. (C) Western blots of myoblasts overexpressing miR-486 or scrambled miRNA controls. Anti-β-tubulin served as a loading control. Western blot analysis of alternately splicing factors SFRS1 and SFRS3 was performed from myotube lysates from which both proteins are most abundantly expressed.