Fig 2.

bmnpv-miR-1 negatively regulates Ran expression by binding to its 3′ UTR in HeLa cells. (A) Luciferase assay showing that bmnpv-miR-1 represses luciferase activity when it binds to the Ran 3′ UTR and that this repression is rescued when the cells are treated with a bmnpv-miR-1-specific antagomir, LNA-1, but the expression remains repressed upon scrambled-LNA treatment. (B) Mutation in the seed region of the bmnpv-miR-1 binding site on the 3′ UTR of Ran mRNA leads to derepression of luciferase activity. (C) To determine the specificity of bmnpv-miR-1–Ran 3′ UTR interaction, a PmirGLO-Ran construct was transfected with an unrelated BmNPV miRNA (bmnpv-miR-3). Also, bmnpv-miR-1 was subjected to an unrelated target site containing the reporter vector (PmirGLO-PPO). In both cases, no significant change was observed in luciferase activity. The assays were done independently three times in triplicate for each sample. Significant differences (P values of <0.05) are denoted by different letters above the error bars. The data are presented as means and standard deviations (SD) (n = 3).