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. 2010 Nov;20(11):1590–1604. doi: 10.1101/gr.107995.110

Figure 5.

Figure 5.

miR-223 is down-regulated with IL15 activation and directly targets the murine GzmB 3′ UTR. (A) Mature miR-223 decreases in abundance in resting NK cells following 24 h of IL15 activation on multiple platforms, including Illumina, SOLiD, microarray, and qRT-PCR. (B) Time course of mature miR-223 down-regulation in mouse NK cells at indicated time points after rmIL15 activation assayed by ABI qRT-PCR assay (N = 3–7 independent experiments). (C) Schema of miR-223 binding site in murine GzmB 3′-UTR sequencing (seed sequence highlighted with bold/underline). Mutated GzmB Δ3′ UTR eliminates the seed binding sites (italics). (D) miR-223 selectively targets the GzmB 3′ UTR. psiCheck2 sensor plasmids containing the GzmB 3′ UTR, Prf1 3′ UTR (negative control), or no 3′ UTR (negative control) were cotransfected in 293T cells with an MND-GFP-miRNA overexpression vector containing a mini-gene of mir-223, mir-21 (negative control), or no miRNA (negative control). Overexpression of miR-223 selectively decreased the luciferase expression compared to no miRNA or miRNA-21 controls. No down-regulation of luciferase was observed for negative control (Prf1 3′ UTR or no 3′ UTR) with miR-223 overexpression. (E) miR-223 targeting of GzmB 3′ UTR is direct. miR-223 down-regulates luciferase controlled by the endogenous GzmB 3′ UTR, but fails to down-regulate luciferase controlled by the GzmB Δ3′ UTR (see B), which lacks the predicted miR-223 seed sequence.