FIGURE 5.

Examination of five potential miR4 target sites. (A) The capability of miR4 to inhibit expression of the transcript of RLuc reporter gene containing one of the five potential miR4 target sites was tested (see Table 1). HP1 indicates hypothetical protein 1; HP2, hypothetical protein 2; ZFD, zinc finger domain; and NEK, NEK kinase. Expression in the absence of miR4 was set at 100% in all the controls. Only the transcript carrying the VSP target site showed an a ∼20% decrease in RLuc activity, suggesting that miR4 specifically regulates VSP gene expression. The results represent the average and standard deviations from at least three independent experiments, and the Students t-test was used to determine P-values. (B) The miR4-mediated repression of Rluc expression from the transcript carrying the VSP target sites was significantly reduced in the GlAgo knockdown cells, indicating that GlAgo is required for miR4 action. (C) Rluc activity was increased by ∼20% when miR4 2′ O-methyl antisense oligo was introduced into the cells, suggesting that endogenous miR4 represses the reporter gene expression. The results represent the average and standard deviations of at least two independent experiments, and the Students t-test was used to determine the P-values.