Figure 6

Centered sites can repress gene expression. (A) Four imperfect centered binding sites for miR-17-5p (left column) and eight for miR-27a (right columns) were tested via luciferase assay. Vertical line, perfect Watson-Crick matches; colon, GU wobble sites; shaded bases, regions outside of the central zone (nucleotides 3 to 15) that may participate in binding; red text, mismatches between the miRNA and its target in the central zone. Note that no other binding sites of any type were predicted within these mRNAs. (B) Mean luciferase activity relative to negative controls from the empty plasmid (pMIR) or pMIR containing one of the imperfect binding sites described above in the 3′ UTR. Asterisks indicate significant reduction in luciferase activity (one-sided t-test; P < 0.05), error bars represent standard error of the mean over three replicates. (C,D) A hybrid AGO2 immunoprecipitation/biotin pull-down was performed as in Figure 2F, and qRT-PCR was used to detect mRNAs confirmed to be targets of miR-17-5p (C) and miR-27a (D) above. Numbering on the x-axis refers to the samples described in Figure 2F. All targets were found to be significantly enriched in the AGO2-enriched fraction (*P ≤ 0.05, **P < 0.01, ***P < 0.001). (E,F) Analysis of luciferase expression levels by qRT-PCR found that only one out of three imperfect centered sites for miR17-5p (E) and one out of four sites for miR-27a (F) showed significantly reduced luciferase mRNA levels (P < 0.05, indicated by asterisks), indicating that most interactions that result in reduced protein are due to translational inhibition and not mRNA degradation.