Differential activities of miR-122 RNA species containing an additional stem bulge. (A) Sequences and predicted structures of the duplex p3 (bulge) and the pre-p3 (bulge) mimetics. The sequence of mature miR-122 is highlighted in bold. The mutated C-nucleotide at position 3 in miR-122 is underlined and marked with an asterisk. The changed cytosines to guanosine nucleotides are boxed. (B) hDicer/TRBP cleavage assays were performed as described in Figure 2A. Samples were incubated with purified hDicer/TRBP for 1 h. (C) Luciferase-based microRNA assays were done as described in Figure 1C. Data include at least three independent replicates; error bars, SEM. (D) GFP-based cleavage assays were performed as described in Figure 2D. Quantitation is shown as the ratio of full-length GFP mRNA to total (full-length plus cleaved) GFP mRNA and represents at least three independent replicates. Error bars, SEM. (E) HCV RNA accumulation assays. Experiments were performed as in Figure 2E. Autoradiographs were quantitated using ImageQuant, and HCV RNA levels were normalized to the amount of γ-actin. Data from cells transfected with miR-122 p3 were set to 100% and used as a positive control. The data shown represent at least three independent replicates. Error bars, SEM.
