FIGURE 6.

CRD-BP counteracts miR-340 action and stabilizes MITF mRNA. A, wild type MITF-expressing plasmid p-BIG-MITF^wt was co-transfected with Tet-off plasmid and the indicated constructs in 451Lu cells. The turnover of MITF transcript was analyzed as Fig. 4E. B, 451Lu cells were transfected with the indicated constructs. 48 h after transfection, the cells were collected and assayed for levels of endogeneous MITF mRNA by quantitative RT-PCR, normalized to GAPDH expression, and presented as percentages of control. C, 451Lu cells were co-transfected with MITF-dependent luciferase-expressing vector pHTRPL4, the indicated shRNA and miR-Sponge-expressing plasmids, and β-galactosidase-expressing plasmid. The values correspond to luciferase activity normalized to β-galactosidase expression. D, construct expressing MITF with both of the miR-340 sites deleted (p-BIG-MITF^δmiR-340) was co-transfected with Tet-off plasmid and the indicated constructs in 451Lu cells. The turnover of MITF transcript was analyzed as Fig. 4E. All of the results are representative of three separate experiments and are expressed as the mean values ± S.D. (error bars). The average half-lives of mRNAs are presented in supplemental Table S3. E, 451Lu and 928 Mel cells were co-transfected with indicated constructs and pTk-Puro. The colonies were selected for puromycin resistance, stained with crystal violet, and counted.