FIG. 1.

Unstable and stable variant β-globin mRNAs can be distinguished in intact cells. (A) Structures of conditionally expressed reporter genes encoding variant β-globin mRNAs. pTRE-β^WT contains the full-length human β-globin gene, including native intronic, exonic, and 3′-flanking sequences (thin, thick, and intermediate gray lines, respectively), downstream of a Tet-conditional TRE promoter (dotted cross-hatching). pTRE-β^ARE104 and pTRE-β^ARE130 are identical to pTRE-β^WT except for a 59-bp ARE instability element (⊠) at either of two 3′UTR positions. (B) Variant β^ARE104 mRNA is unstable in cultured cells. The levels of β^WT and β^ARE104 mRNAs in transiently transfected HeLa^tTA cells were assessed by RT-PCR⁺¹ at defined intervals following Dox exposure. The intensities of the β^WT bands were balanced by adjusting sample loading. C1 and C2 contain RNA from cells transfected singly with pTRE-β^WT and pTRE-β^ARE104, respectively. (C) ARE-mediated destabilization of β-globin mRNA in cultured cells. The autoradiograph in panel B was analyzed by PhosphorImager densitometry and the β^ARE104/β^WT ratio plotted (black squares). Results from a parallel study of β^ARE130 are also plotted (gray circles). The dashed line indicates the β^ARE/β^WT ratio that would be observed if the two mRNAs were equally stable.