Figure 8: CUGBP1 binds the 3′UTR of MICB at a GU-rich element and regulates its expression.

(a) The predicted binding sequence of CUGBP1 at position 616-661 in the 3′UTR of MICB (top, bold black letters). Sequence of the mutation in the CUGBP1 predicted binding site, named GUmut (bottom). Mutated nucleotides are indicated in red. (b) RKO cells were transfected with one of two reporters: fused to the WT 3′UTR of MICB and to the GUmut 3′UTR. Forty-eight hours post transfection, Firefly activity was measured relative to the Renilla luciferase. Activity of the reporters was calculated relatively to the activity of the reporter fused to the WT 3′UTR. Shown is relative averaged mean±s.d. of combined three independent experiments. *P<0.002, by Student’s t-test. (c) HEK293T cells were transduced with lentiviruses encoding MICB–CDS fused either to a MICB WT 3′UTR or to the GUmut 3′UTR. The cells were then transduced either with a specific shCUGBP1 shRNA (black histograms) or a control-shRNA (grey empty histograms). MICB (top) and MICA (bottom) expressions were evaluated by FACS. Filled grey histograms represent background staining. Data are a representative of four independent experiments. (d) Quantification of all FACS experiments performed in (c). Shown is the relative average MICB MFI±s.d. of all experiments. *P<0.02 by Student’s t-test. (e) NK killing assay was performed on HEK293T cells transduced either with MICB–CDS fused to MICB WT 3′UTR or transduced with MICB–CDS fused to GUmut 3′UTR. These cells were then transduced either with a control-shRNA or with a specific shCUGBP1. Shown is relative average killing±s.d. of three independent experiments, calculated relative to the specific cells transduced with a control-shRNA. *P<0.005, by Student’s t-test.