Effect of CPB2 SNPs on mRNA stability. (A) The parental construct consists of the CPB2 3′-UTR (nucleotides +1273 to +1819) inserted into pC7βG downstream of the rabbit β-globin cDNA.³³  The construct also contains the cytomegalovirus promoter (P_CMV), translation termination cassette (TTC), and simian virus 40 polyadenylation sequences (SV40 PA). Using site-directed mutagenesis, SNPs in the CPB2 3′-UTR, at positions +1344, +1542, and +1583, were introduced either alone or in the haplotypic combinations observed in the human population. The H1 to H4 haplotypes are those described by Henry and coworkers.²⁷  The haplotypes denoted H1^† to H4^† contain the +1344 A substitution. Also shown are the locations of the 3 polyadenylation signal sequences present in the 3′-UTR at +1660, +1693, and +1819. (B) Representative Northern blots for each construct are shown, along with a representative blot for the parental pC7βG plasmid (βG). In each case, the lanes correspond to mRNA harvested at 0, 1, 2, 4, 6, and 8 hours after the addition of actinomycin D. The full collection of blots is available as Figure S1 (available on the Blood website; see the Supplemental Materials link at the top of the online article). (C) HepG2 cells stably transfected with the β-globin fusion mRNA reporter plasmids indicated to the right of the graph, or with a reporter plasmid lacking CPB2 3′-UTR sequences (βG), were treated with actinomycin D. RNA was harvested at various times after the addition of the drug and subjected to Northern blot analysis using a probe specific for rabbit β-globin. To control for differences in RNA loading and transfer, the blots were stripped and hybridized with a probe specific for human GAPDH. Corrected fusion transcript abundance was normalized to that observed before the addition of actinomycin D to obtain the decay curves shown. The data shown are the means plus or minus SEM of 3 independent experiments. The mean half-lives of the respective fusion transcripts are shown to the right of the graph.