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. 2012 Nov 29;8(11):e1003105. doi: 10.1371/journal.pgen.1003105

Figure 5. Uridylation of select miRNAs relieves silencing of the IGF-1 3′ UTR.

Figure 5

To assess miRNA targeting of IGF-1 the firefly luciferase reporter carrying the IGF-1 3′-UTR and control Renilla Luciferase (for normalization) were co-transfected into H1299 cells with various miRNA mimetics. (A) Mimetics for miR-126-5p, miR-194-2-3p and miR-379 effectively targeted the IGF-1 3′-UTR, while Let-7d did not. *p<0.05 vs. control, N = 3, by one-way ANOVA. (B) Mimetics of miR-194-2-3p, miR-126-5p, and miR-379 with or without 2 3′ uridine residues were used to assess the impact of uridylation on the effect of silencing by these miRNAs, revealing that uridylation diminished silencing by miR-126-5p and miR-379 but not miR-194-2-3p. *p<0.05 vs. mature by two-way ANOVA by student's t-test, N = 4. (C–D) miR-126-5p or miR-379 mimetics bearing 0, 1, 2, or 4 terminal uridines show a maximal effect on miRNA silencing by a single uridine addition. Sequences reflect those identified in sequencing libraries. *p<0.05 vs. control by one-way ANOVA, N = 4. (E) Unlike terminal uridylation, mutation of two bases in the seed sequence of miR-126-5p completely reversed silencing of the IGF-1 3′-UTR. (F) Non-uridylated or dual-uridylated mimetics of miR-126-5p and miR-379 illustrate the combinatorial role of multiple uridylated miRNAs targeting the same transcript. *p<0.05 vs control by one-way ANOVA, N = 4.