FIGURE 3.

The CURE and CARE represent separate cis-acting elements that differentially regulate the inhibitory activity of the CD154 3′-UTR. a, the murine CD154 3′-UTR limits pcDNA 3.1 reporter gene expression in transiently transfected HeLa cells at the level of cytoplasmic poly(A) mRNA as measured by oligo(dT)-primed RT-PCR. Deletion of both CURE and CARE (CUCARE-) was required to eliminate inhibitory effect of CD154 3′-UTR on poly(A) mRNA (n = 6). p values are shown relative to that seen with luciferase controls. b, the CURE and CARE alone are sufficient to limit cytoplasmic poly(A) mRNA accumulation (n = 6). c and d, pTRE-based plasmids were transiently transfected into HeLa Tet-Off cells with transcriptional inhibition triggered by the addition of doxycycline (1 μg/ml). Cytoplasmic RNA was extracted at the time of doxycycline addition and specified times thereafter with poly(A) RNA purified and analyzed by oligo(dT)-primed RT-PCR. The level of input RNA measured at time 0 for each vector is assigned a value of 100%, although both the CURE- and CARE-vectors reduced mRNA levels. Increased mRNA decay rates are only seen with reporters (c, CD154, CARE-, and CURE+) containing the CURE in their 3′-UTR. Data shown are representative of three experiments each.