miR-181 targets genes with multiple coding-region sites. (A) Response of mRNAs to the introduction of miR-181a into HeLa cells. Plotted are cumulative distributions of fold-changes for mRNAs with the indicated numbers and types of sites. Except for the two categories indicated, categories with ORF sites excluded mRNAs with 3′ UTR sites, and those with 3′ UTR sites excluded mRNAs with ORF sites. (B) Mean fold-changes for the categories of A. Error bars show standard deviation from bootstrapping. (C) The propensity of miR-181 to have many ORF sites. Plotted are numbers of genes containing at least the indicated number of sites for either miR-181 (green) or the median across all miRNAs (blue). Error bars show the interquartile range. (D) miR-181a-mediated repression of reporters with miR-181 ORF sites. Reporters included the luciferase ORF following the ORF of the indicated mRNA. Fold repression was calculated relative to that of the noncognate miRNA, miR-23a (see Methods). Plotted are the normalized values, with error bars representing the third largest and third smallest values (n = 12; p < 10−6, except for the control, luciferase-only reporter, for which p = 0.32). (E) Dependence of ZNF20 repression on miR-181 ORF sites. Repression was calculated and depicted as in D, additionally normalizing repression of the reporter with wild-type sites (WT) to that of a reporter in which the eight ORF sites were mutated (n = 12; p < 10−6). (F) Direct repression of RBAK ORF and 3′ UTR mediated by miR-181a, as assayed by luciferase assays. Repression was calculated and depicted as in D, additionally normalizing repression of the reporter with wild-type sites (WT) to that of the mutant reporter in which the 19 ORF or two 3′ UTR sites were mutated (n = 12; p = 7 × 10−7 and p = 0.0009, respectively).
