Abstract
BackgroundThe normal accumulation of adult α and β globins in definitive erythrocytes is critically dependent upon processes that ensure that the cognate mRNAs are maintained at high levels in transcriptionally silent, but translationally active progenitor cells. The impact of these post-transcriptional regulatory events on the expression of embryonic ζ globin is not known, as its encoding mRNA is not normally transcribed during adult erythropoiesis. Recently, though, ζ globin has been recognized as a potential therapeutic for α thalassemia and sickle-cell disease, raising practical questions about constitutive post-transcriptional processes that may enhance, or possibly prohibit, the expression of exogenous or derepresssed endogenous ζ-globin genes in definitive erythroid progenitors.MethodsThe present study assesses mRNA half-life in intact cells using a pulse-chase approach; identifies cis-acting determinants of ζ-globin mRNA stability using a saturation mutagenesis strategy; establishes critical 3′UTR secondary structures using an in vitro enzymatic mapping method; and identifies trans-acting effector factors using an affinity chromatographical procedure.ResultsWe specify a tetranucleotide 3′UTR motif that is required for the high-level accumulation of ζ-globin transcripts in cultured cells, and formally demonstrate that it prolongs their cytoplasmic half-lives. Surprisingly, the ζ-globin mRNA stability motif does not function autonomously, predicting an activity that is subject to structural constraints that we subsequently specify. Additional studies demonstrate that the ζ-globin mRNA stability motif is targeted by AUF1, a ubiquitous RNA-binding protein that enhances the half-life of adult β-globin mRNA, suggesting commonalities in post-transcriptional processes that regulate globin transcripts at all stages of mammalian development.ConclusionsThese data demonstrate a mechanism for ζ-globin mRNA stability that exists in definitive erythropoiesis and is available for therapeutic manipulation in α thalassemia and sickle-cell disease.
Highlights
The normal accumulation of adult α and β globins in definitive erythrocytes is critically dependent upon processes that ensure that the cognate mRNAs are maintained at high levels in transcriptionally silent, but translationally active progenitor cells
Embryonic α-like ζ globin is produced during the first gestational trimester in primitive erythroblasts that originate in blood islands of the yolk sac, while fetal/adult α globin is induced at the end of this interval and continues to express in definitive erythroblasts that are initially produced in the liver and, subsequently, in the bone marrow [1,2]
A transcriptional chase strategy identifies cis-acting regulatory determinants within the ζ-globin 3′ region of untranslated mRNA (3′UTR) Transgenic human ζ globin accumulates to high levels in mouse erythrocytes, consistent with the existence of cisacting determinants that stabilize the cognate ζ-globin mRNA in transcriptionally silent progenitor cells [6,7,18]
Summary
The normal accumulation of adult α and β globins in definitive erythrocytes is critically dependent upon processes that ensure that the cognate mRNAs are maintained at high levels in transcriptionally silent, but translationally active progenitor cells. When compelled to express in definitive erythroid cells, embryonic ζ globin assembles with adult β globin into heterotetrameric Hb ζ2β2 (Hb Portland-2), which exhibits O2-binding and allosteric properties that differ modestly from those of Hb α2β2 (Hb A, the principal adult hemoglobin), but that remain fully compatible with normal adult physiology [8,9] The significance of this approach for treating αglobin deficiency states (α thalassemia) is illustrated by evidence that transgenic human ζ globin fully reverts the pathological phenotype of mice containing heterozygous knockout of their endogenous α-globin genes and, remarkably, restores full viability to animals with homozygous, embryonic-lethal inactivation of these same genes [6]. These two developing applications for ζ globin–effected through either gene-reactivation or gene-supplementation strategies– recommend careful description of the events that contribute to its regulation in definitive erythroid cells
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