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. 2012 Oct 26;287(51):43007–43018. doi: 10.1074/jbc.M112.386938

FIGURE 9.

FIGURE 9.

USP15 regulates CRAF at a post-transcriptional level. A, CRAF mRNA from HeLa cells treated for 24 h with two individual oligos targeting USP15 was assessed by RT-PCR using two primer sets (CRAF and CRAFe/e) designed to amplify different regions of the CRAF transcript. Values from four biological replicates were normalized to actin mRNA (error bars, S.D. two-tailed t test compared with control; CRAF-USP15-1, p < 0.00025; USP15-2, p < 0.05; CRAFe/e-USP15-1, p < 0.0005). B, USP15 depletion does not decrease the transcriptional activity of a minimal CRAF promoter. HeLa cells were cotransfected with two individual siRNAs targeting USP15 (USP15-1, USP15-2) or a control siRNA (Control) together with a plasmid expressing firefly luciferase under the control of a SV40 promoter (pGL3-Control) or a minimal CRAF promoter (pGL3-CRAFpr) and the Renilla luciferase reporter construct, phRL-tk. Cells were lysed 48 h post-transfection and analyzed using the Promega dual-luciferase reporter assay system. Results from six biological replicates are shown normalized to phRL-tk reporter activity (n = 6, error bars, S.D.). C, USP15 depletion does not markedly alter the amount of CRAF pre-mRNA. Randomly primed cDNA, derived from RNA extracted from HeLa cells treated for 24 h with control reagent or two individual siRNA oligos targeting USP15 (USP15-1, USP15-2), or USP4, was analyzed by quantitative real-time RT-PCR. Primers were designed to anneal to exon junction sequences (USP15, CRAF e/e) or exon-intron junctions (CRAF e/i) to assess mature and pre-mRNA, respectively. Values from three biological replicates were normalized to actin mRNA. Two-tailed t test; USP15-USP15-1, p < 0.005; USP15-2, p < 0.025; CRAFe/e-USP15-1, p < 0.01; USP15-2, p < 0.05. D, HeLa cells were treated for 48 h with individual siRNAs targeting USP15 (USP15-1, USP15-2) or a control siRNA (Control) before transfection for another 24 h with plasmids expressing firefly luciferase under the control of a minimal SV40 promoter with or without the appendage of the 3′-UTR of CRAF. Cells were cotransfected with phRL-tk. Comparison of the relative luciferase activity from the basic and pGL3-CRAF-3′-UTR plasmids suggests that USP15 depletion has a destabilizing effect on the 3′-UTR-harboring transcript. Data are of three biological replicates. Error bars, S.D., paired two-tailed t test for pGL3-CRAF-UTR compared with pGL3-Control, USP15-1 and USP15-2, p < 0.0001.