The stability of hϵ-globin mRNA is dictated by determinants within its 3′ UTR. (A) Structures of doxycycline-conditional genes encoding hϵ- and hβ-globin mRNAs with reciprocal exchange of their 3′UTRs. TRE-hϵ^3′β and TRE-hβ^3′ϵ are identical to parental TRE-hϵ and TRE-hβ except for nucleotide-specific reciprocal exchange of their 3′UTRs. Structural elements derived from the hϵ- and hβ-globin genes are indicated in gray and black, respectively. The tTA-responsive TRE transcriptional control element is diagonally shaded. (B) Representative transcriptional chase analysis of chimeric globin mRNAs in erythroid cells. tTA-expressing MEL cells were cotransfected with TRE-hϵ^3′β and TRE-hβ^3′ϵ, and RNAs collected at defined intervals after doxycycline exposure were assessed by a 2-probe RNase protection method. Linearity controls have been cropped to preserve image clarity. (C) hϵ^3′β and hβ^3′ϵ are equally stable in erythroid MEL cells. The study described in panel B was performed in triplicate. The hϵ^3′β/hβ^3′ϵ band intensities at defined intervals after doxycycline exposure were determined by PhosphorImager densitometry and average values plotted. The relative stabilities of the parental hϵ/hβ mRNAs have been reproduced from Figure 1 for comparison (gray line). Error bars indicate 1 SD. (D) Representative transcriptional chase analysis of chimeric hϵ/hβ-globin mRNAs in nonerythroid cells. tTA-expressing HeLa cells that were cotransfected with TRE-hϵ^3′β and TRE-hβ^3′ϵ were analyzed as described in panel B. (E) The hϵ^3′β-globin and hβ^3′ϵ-globin mRNAs are equally stable in nonerythroid HeLa cells. The study described in panel D was performed in triplicate and average band intensities plotted. A gray line indicates the relative stabilities of the parental hϵ/hβ mRNAs previously established in Figure 1. Error bars indicate 1 SD. (F) The relative stabilities of hϵ- and hβ-globin mRNAs in intact erythroid cells in vivo are dependent on elements within their 3′UTRs. The stabilities of hϵ^3′β-globin and hβ^3′ϵ-globin mRNAs were determined in 3 or more mice from each of 2 hβ^3′ϵ and 4 hϵ^3′β transgenic mouse lines (identified at bottom) using marrow-reticulocyte analysis. Vertical arrows indicate the difference between the average stabilities of the chimeric mRNAs (arrowhead) and the stabilities of hβ- and hϵ-globin mRNAs containing their native 3′UTRs (arrow tail).