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. 2017 Jul 26;45(17):10218–10228. doi: 10.1093/nar/gkx640

Figure 2.

Figure 2.

Superior regulation of CNTFR and TMEM2 3′-UTRs is mediated by strong miRNA binding to single target sites. (A) Predicted hybridization of the binding sites of selected targets. (Mutant and modified sites are depicted in Supplementary Figure S4B). (B) Results of the dual-fluorescent reporter assays. Measurements with the antimiRs LNA-21 (n = 8) or LNA-let-7 family (n = 5) were normalized to LNA-Ctrl. Depicted are values from cells in the bin with 100–200 AFU RFP values (as in Figure 1D). (C) Results of the competitive binding assay. Dual fluorescent reporters specific for miR-21 or let-7 activity (binding sites are depicted in Supplementary Figure S2) were co-transfected with the analyzed UTRs inserted 3′ of iRFP to compare their potencies as inhibitors of the respective miRNAs. Transfection ratios of reporter to inhibitor were 1:1 for miR-21 (n = 4) and 1:10 for let-7 (n = 7) targets. Depicted is the bin with 400–800 AFU for miR-21 and 100–200 AFU for let-7. (D and E) Design and results of the Ago2-RIP assay to compare changes in RIP-fraction upon seed mutation (n = 5). *P < 0.05 in ANOVA for repeated measures with a post-hoc Tukey’s test.