FIGURE 2.

The murine CD154 3′-UTR has two cis-acting elements that regulate reporter gene expression cytoplasmic poly(A)⁺ mRNA accumulation. a, murine CD154 3′-UTR limits pcDNA 3.1 reporter gene expression in transiently transfected HeLa cells. Deletion of both CURE and CARE (CUCARE-) was required to eliminate inhibitory effect of CD154 3′-UTR on luciferase gene expression. Data shown are an average of four experiments, with error bars representing the S.E. (n = 4). b, the CARE and CURE function to regulate reporter gene expression in human peripheral blood mononuclear cells under both resting and activated conditions. Transient transfection with the specified reporter vectors was performed, and luciferase activity was measured under basal (black bars) or activated (open bars) conditions, where phorbol 12-myristate 13-acetate (10 ng/ml) and ionomycin (1 μm) were added for the last 4 h. c, mutations of the polycytidine or ARE in the context of the CUCARE-deletions had no effect on gene expression. HeLa cells were transiently transfected with pcDNA 3.1-based plasmids containing the control, CD154 3′-UTR, or CUCARE-, mutation. The CUCARE-polyCmut and the CUCARE-AREmut additionally contained deletions of the polycytidine and ARE sequences, respectively. Error bars are not visible at this scale in this representative experiment (n = 4). d, the CURE and CARE alone are each sufficient to limit reporter gene expression effect on luciferase expression, whereas combination of the CURE and CARE (CUCARE+) did not enhance their activity (n = 4).