FIGURE 5.

The presence of the 3′-UTR-CARE is associated with a shortened poly(A) tail but increased cytoplasmic mRNA stability. a, the presence of the 3′-UTR-CARE is associated with increased deadenylation of reporter gene as well as native CD154 mRNA. Cytoplasmic RNA extracts from transiently transfected HeLa cells with specified pcDNA 3.1-based plasmids were assayed for poly(A) tail length. Cytoplasmic luciferase mRNA from transient transfected HeLa cells containing the 3′-UTR CARE has a markedly shortened (<50-nt) poly(A) tail relative to control or 3′-UTR CURE as measured by the LM-PAT assay. The arrow indicates the predicted length of fully deadenylated fragments. Note the extended poly(A) tail length (∼150 A bases) from transfections with control and CURE constructs. Presence of CURE markedly reduced luciferase mRNA while not affecting poly(A) length; the data shown in this gel have twice as much PCR product in this lane loaded to make this product visible by EtBr staining. The presence of the CURE and CARE (CUCA+) yielded a short poly(A) tail as well. b, the 3′-UTR CARE results in decreased luciferase mRNA poly(A) tail length in nuclear, cytoplasmic, and polysomal fractions. Right-hand arrows show the predicted fragments following MnlI restriction enzyme digestion, establishing the identity of the amplified product. c, the presence of the CARE in the 3′-UTR resulted in increased mRNA stability, whether measured by RT-PCR following oligo(dT)- or RH-dependent priming.