Figure 4.

LGP2 represses pre-miRNA recruitment by TRBP to inhibit microRNA (miRNA) maturation. (A) Schematic diagram of the pre-miRNA-mediated RNA silencing pathway. TRBP recruits pre-miRNA into Dicer to promote miRNA maturation. The mature miRNA is loaded on Argonaute (AGO) in the RNA-induced silencing complex (RISC), and represses target RNA with sequence complementarity. (B) Immunoprecipitation of TRBP and Northern blot of pre-miRNA targeting firefly luciferase (pre-miLuc). Plasmids encoding FLAG-TRBP, pre-miLuc, and LGP2-Myc were co-transfected to HeLa cells and immunoprecipitation was performed with anti-FLAG-antibody. Total RNA purified from each immunoprecipitate was subjected to Northern blot analysis to detect pre-miLuc. U6 was used as a control. (C) Northern blots of mature miLuc in WT, TRBP^−/−, or LGP2^−/− cells with or without IFN. Plasmids encoding pre-miLuc were transfected into WT, TRBP^−/− or LGP2^−/− cells in the absence and presence of IFN. The bar graph shows the quantified signal intensities of mature miLuc relative to the levels in HeLa-WT cells without IFN (mature RNA level of 1). The experiments were performed in triplicate and P-values were determined by Student's t-test (*P < 0.05). (D) RNA silencing activity assay using dual luciferase reporter assay in WT, TRBP^−/− or LGP2^−/− cells. Plasmids encoding pre-miLuc, firefly luciferase, and Renilla luciferase were co-transfected into WT, TRBP^−/− or LGP2^−/− cells, and relative luciferase activity was measured. The experiments were performed in triplicate and P-values were determined by Student's t-test (*P < 0.05).