Figure 4

Mutations in the recognition elements of miR-146a*, miR-524-5p and miR-582-3p reveal a direct regulation of the miRNA on luciferase transcripts coupled to the NFL 3′UTR. (A) Mutants of MREs within the NFL 3′UTR. Only the first two nucleotides of the 3′ end of each MRE were mutated (arrows) preventing broad changes in the NFL 3′UTR secondary structure. (B) ΔGs of wild type (WT) and mutant (Mut) MREs within NFL 3′UTR. (C) Reporter gene assay performed with mutant NFL 3′UTRs. The results of firefly luciferase activity were normalized with Renilla reporter luciferase activity and the effect of each miRNA on the luciferase mRNA alone (pmirGLO vector control). Data are expressed as relative change and show up-regulation as positive values and down-regulation as negative values. Experiments were performed in triplicate. Result are shown as mean ± SEM (t-test: *** = p < 0.001; * = p < 0.05, relative to the pmirGLO vector control). All the NFL 3′UTR mutants showed a significant decrease in the effect of each miRNA compared with the wild type.