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Hb Baden (β18Val→Met) is a rare variant hemoglobin that has never been functionally or clinically characterized. We describe a Hb Baden heterozygote who exhibits normal growth and development, as well as age- and gender-appropriate hematological parameters. Surprisingly, in vitro analyses demonstrate that Hb Baden is relatively unstable and exhibits an abnormally high affinity for O2. These properties are likely to affect the physiologies of individuals who inherit the βBaden mutation in trans to a determinant for either a functionally relevant hemoglobin-opathy or a mild thalassemia. The data also provide insights into the function of the AB-segment/A-helix of the β-globin, supporting a structural model in which this poorly understood region serves as a scaffold that fixes the positions of other helices that directly impact β-globin function.

A previously unrecognized function of normal human hemoglobins occurring during protein assembly is described, i.e. self-regulation of subunit pairings and their durations arising from the variable strengths of their subunit interactions. Although many mutant human hemoglobins are known to have altered subunit interface strengths, those of the normal embryonic, fetal, and adult human hemoglobins have not been considered to differ significantly. However, in a comprehensive study of both types of subunit interfaces of seven of the eight normal oxy human hemoglobins, we found that the strengths, i.e., the free energies of the tetramer-dimer interfaces, contrary to previous reports, differ by 3 orders of magnitude and display an undulating profile similar to the transitions ("switches") of various globin subunit types over time. The dimer interface strengths are also variable and correlate linearly with their developmental profile. Embryonic hemoglobins are the weakest; fetal hemoglobin is of intermediate strength, and adult hemoglobins are the strongest. The pattern also correlates generally with their different O(2) affinities and responses to allosteric regulatory molecules. Acetylation of fetal hemoglobin weakens its unusually strong subunit interactions and occurs progressively as its level of expression diminishes and adult hemoglobin A formation begins; a causal relationship is suggested. The relative contributions of globin gene order and competition among subunits due to differences in their interface strengths were found to be complementary and establish a connection among genetics, thermodynamics, and development.

OBJECTIVES The expression of human gamma globin is developmentally regulated through mechanisms that affect the transcriptional activity of its encoding gene. The current manuscript investigates whether the efficiency of this process might be enhanced though an unrecognized post-transcriptional event that defines the stability of gamma-globin mRNA. METHODS Experiments were conducted in vivo in transgenic mice expressing human gamma globin in their adult erythroid cells. The expression of gamma-globin protein was manipulated by breeding the transgene into animals producing different levels of endogenous mouse beta-globin. Changes in the expression of gamma globin were then correlated to measures of gamma-globin mRNA stability in vivo. RESULTS Human gamma globin was expressed at higher levels in thalassemic than in than non-thalassemic control transgenics, paralleling a highly significant increase in the stability of gamma-globin mRNA. Other molecular events-including possible transcriptional induction of the transgene, or an increase in the stability of the gamma-globin protein-did not appear to contribute to the observed increase in transgene expression. As anticipated, the stability of gamma-globin mRNA also fell in bitransgenic animals that co-expressed human beta-globin mRNA. CONCLUSIONS Our results are consistent with a model for dynamic post-transcriptional control of gamma-globin gene expression, through modulation of the stability of its encoding mRNA. Moreover, the stability of gamma-globin mRNA appears to be inversely related to ambient levels of co-expressed beta-globin mRNA. This data suggests that therapeutic gene-reactivation and/or gene-replacement therapies may be particularly effective in individuals with severe forms of beta-thalassemia.

Functional studies of embryonic epsilon-globin indicate that individuals with beta thalassemia or sickle cell disease are likely to benefit from therapeutic, transcriptional derepression of its encoding gene. The success of epsilon-globin gene-reactivation strategies, however, will be tempered by the stability that epsilon-globin mRNA exhibits in developmental stage-discordant definitive erythroid progenitors. Using cell culture and transgenic mouse model systems, we demonstrate that epsilon-globin mRNA is modestly unstable in immature, transcriptionally active erythroid cells, but that this characteristic has relatively little impact on the accumulation of epsilon-globin mRNA at subsequent stages of terminal differentiation. Importantly, the constitutive stability of epsilon-globin mRNA increases in transgenic mouse models of beta thalassemia, suggesting that epsilon- and beta-globin mRNAs are coregulated through a shared posttranscriptional mechanism. As anticipated, relevant cis-acting determinants of epsilon-globin mRNA stability map to its 3' UTR, consistent with the positioning of functionally related elements in other globin mRNAs. These studies demonstrate that posttranscriptional processes do not pose a significant practical barrier to epsilon-globin gene reactivation and, moreover, indicate that related therapeutic strategies may be particularly effective in individuals carrying beta-thalassemic gene defects.

Hemoglobin ζ 2 β 2 S is generated by substituting embryonic ζ-globin subunits for the normal α-globin components of Hb S ( α 2 β 2 S ) . This novel hemoglobin has recently been shown to inhibit polymerization of Hb S in vitro and to normalize the pathological phenotype of mouse models of sickle cell disease in vivo. Despite its promise as a therapeutic tool in human disease, however, the basic O2-transport properties of Hb ζ 2 β 2 S have not yet been described. Using human hemoglobins purified from complex transgenic-knockout mice, we show that Hb ζ 2 β 2 S exhibits an O2 affinity as well as a Hill coefficient, Bohr response, and allosteric properties in vitro that are suboptimally suited for physiological O2 transport in vivo. These data are substantiated by in situ analyses demonstrating an increase in the O2 affinity of intact erythrocytes from mice that express Hb ζ 2 β 2 S . Surprisingly, though, co-expression of Hb ζ 2 β 2 S leads to a substantial improvement in the tissue oxygenation of mice that model sickle cell disease. These analyses suggest that, in the context of sickle cell disease, the beneficial antisickling effects of Hb ζ 2 β 2 S outweigh its O2-transport liabilities. The potential structural bases for the antisickling properties of Hb ζ 2 β 2 S are discussed in the context of these new observations.

Determinants of mRNA stability are frequently positioned in the 3'UTR where they are not subject to disruption by actively translating ribosomes. Two related individuals with beta thalassaemia who carry a beta-globin gene containing a 13 nt deletion in its 3'UTR have recently been described. Its position within the 3'UTR, as well as its relative distance from other known functionally important elements, suggested that the deletion might overlay previously unrecognized determinants of beta-globin mRNA stability. We studied the impact of the Delta13 mutation on beta-globin gene expression in vitro and in vivo. The adverse effect of the Delta13 mutation on beta-globin expression was confirmed in studies utilizing reticulocytes from a betaDelta13 heterozygote, which indicated a sixfold reduction in the relative level of the mutant mRNA. Additional in vitro analysis indicated that the deletion did not affect the capacity of the betaDelta13 mRNA to assemble an mRNA-stabilizing mRNP 'beta-complex'. Unexpectedly, functional tests in both primary erythroid cells and in a transgenic mouse model demonstrated that the betaDelta13 mRNA was fully stable, suggesting that the Delta13 mutation affects accumulation of the fully processed mRNA at an earlier step. Consistent with this, there was a relative excess of unprocessed betaDelta13 mRNA in erythroid progenitors from a betaDelta13 heterozygote. Taken together, these results define a new thalassaemic determinant, which acts to decrease beta-globin mRNA levels by inhibiting the efficiency of nuclear processing events, and suggest a previously unanticipated complexity to the role of the 3'UTR elements in the regulation of beta-globin gene expression.

Background 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. 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. Methods The 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. Results We 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. Conclusions These 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.

Embryonic ζ- and ε-globin subunits assemble with each other and with adult α- and β-globin subunits into hemoglobin heterotetramers in both primitive and definitive erythrocytes. The properties of these hemoglobins—Hbs Gower-1 (ζ2ε2), Gower-2 (α2ε2), and Portland-2 (ζ2β2)—have been incompletely described as they are difficult to obtain in quantity from either primary human tissue or conventional expression systems. The generation of complex transgenic–knockout mice that express these hemoglobins at levels between 24% and 70% is described, as are efficient methods for their purification from mouse hemolysates. Key physiological characteristics—including P50, Hill coefficient, Bohr effect, and affinity for 2,3-BPG—were established for each of the 3 human hemoglobins. The stability of each hemoglobin in the face of mechanical, thermal, and chemical stresses was also determined. Analyses indicate that the ζ-for-α exchange distinguishing Hb Portland-2 and Hb A alters hemoglobin O2-transport capacity by increasing its P50 and decreasing its Bohr effect. By comparison, the ε-for-β exchange distinguishing Hb Gower-2 and Hb A has little impact on these same functional parameters. Hb Gower-1, assembled entirely from embryonic subunits, displays an elevated P50 level, a reduced Bohr effect, and increased 2,3-BPG binding compared to Hb A. The data support the hypothesis that Hb Gower-2, assembled from reactivated ε globin in individuals with defined hemoglobinopathies and thalassemias, would serve as a physiologically acceptable substitute for deficient or dysfunctional Hb A. In addition, the unexpected properties of Hb Gower-1 call into question a common hypothesis for its primary role in embryonic development.

The developmental stage-specific expression of human globin proteins is characterized by a switch from the coexpression of zeta- and alpha-globin in the embryonic yolk sac to exclusive expression of alpha-globin during fetal and adult life. Recent studies with transgenic mice demonstrate that in addition to transcriptional control elements, full developmental silencing of the human zeta-globin gene requires elements encoded within the transcribed region. In the current work, we establish that these latter elements operate posttranscriptionally by reducing the relative stability of zeta-globin mRNA. Using a transgenic mouse model system, we demonstrate that human zeta-globin mRNA is unstable in adult erythroid cells relative to the highly stable human alpha-globin mRNA. A critical determinant of the difference between alpha- and zeta-globin mRNA stability is mapped by in vivo expression studies to their respective 3' untranslated regions (3'UTRs). In vitro messenger ribonucleoprotein (mRNP) assembly assays demonstrate that the alpha- and zeta-globin 3'UTRs assemble a previously described mRNP stability-determining complex, the alpha-complex, with distinctly different affinities. The diminished efficiency of alpha-complex assembly on the zeta 3'UTR results from a single C-->G nucleotide substitution in a crucial polypyrimidine tract contained by both the human alpha- and zeta-globin mRNA 3'UTRs. A potential pathway for accelerated zeta-globin mRNA decay is suggested by the observation that its 3'UTR encodes a shortened poly(A) tail. Based upon these data, we propose a model for zeta-globin gene silencing in fetal and adult erythroid cells in which posttranscriptional controls play a central role by providing for accelerated clearance of zeta-globin transcripts.

Therapeutic regulation of globin genes is a primary goal of translational research aimed toward hemoglobinopathies. Signal transduction was used to identify chromatin modifications and transcription factor expression patterns that are associated with globin gene regulation. Histone modification and transcriptome profiling were performed using adult primary CD34+ cells cultured with cytokine combinations that produced low versus high levels of gamma-globin mRNA and fetal hemoglobin (HbF). Embryonic, fetal, and adult globin transcript and protein expression patterns were determined for comparison. Chromatin immunoprecipitation assays revealed RNA polymerase II occupancy and histone tail modifications consistent with transcriptional activation only in the high-HbF culture condition. Transcriptome profiling studies demonstrated reproducible changes in expression of nuclear transcription factors associated with high HbF. Among the 13 genes that demonstrated differential transcript levels, 8 demonstrated nuclear protein expression levels that were significantly changed by cytokine signal transduction. Five of the 8 genes are recognized regulators of erythropoiesis or globin genes (MAFF, ID2, HHEX, SOX6, and EGR1). Thus, cytokine-mediated signal transduction in adult erythroid cells causes significant changes in the pattern of globin gene and protein expression that are associated with distinct histone modifications as well as nuclear reprogramming of erythroid transcription factors.

Gene replacement or gene reactivation therapies for sickle-cell disease (SCD) typically target the mutant beta(S)-globin subunits of hemoglobin-S (alpha(2)beta(S)(2)) for substitution by nonpathological beta-like globins. Here we show, in vitro and in vivo in a transgenic mouse model of SCD, that the adverse properties of hemoglobin-S can be reversed by exchanging its normal alpha-globin subunits for zeta-globin, an endogenous, developmentally silenced, non-beta-like globin.

The development of red blood cells (erythrocytes) is distinguished by high-level production of the oxygen carrier, haemoglobin A (HbA), a heterotetramer of alpha- and beta-haemoglobin subunits. HbA synthesis is coordinated to minimize the accumulation of free subunits that form cytotoxic precipitates. Molecular chaperones that regulate globin subunit stability, folding or assembly have been proposed to exist but have never been identified. Here we identify a protein stabilizing free alpha-haemoglobin by using a screen for genes induced by the essential erythroid transcription factor GATA-1 (refs 4, 5). Alpha Haemoglobin Stabilizing Protein (AHSP) is an abundant, erythroid-specific protein that forms a stable complex with free alpha-haemoglobin but not with beta-haemoglobin or haemoglobin A (alpha(2)beta(2)). Moreover, AHSP specifically protects free alpha-haemoglobin from precipitation in solution and in live cells. AHSP-gene-ablated mice exhibit reticulocytosis and abnormal erythrocyte morphology with intracellular inclusion bodies that stain positively for denatured haemoglobins. Hence, AHSP is required for normal erythropoiesis, probably acting to block the deleterious effects of free alpha-haemoglobin precipitation. Accordingly, AHSP gene dosage is predicted to modulate pathological states of alpha-haemoglobin excess, such as beta-thalassaemia.

Important therapeutic approaches to sickle-cell disease (SCD) are based upon the observation that the abnormal properties of Hb S (Hb α2βS2) can be mitigated by exchanging the pathological βS-globin subunit for a related β-like subunit. We previously demonstrated that exchange of the non-pathological α-globin subunit for a ζ-globin subunit (a developmentally silenced globin that can be derepressed both by natural and experimental conditions) inhibits deoxyHb S polymer assembly in vitro and reverses hematological, biochemical, and physiological characteristics of SCD in mouse models in vivo. While its therapeutic potential is clear, the underlying structural basis for the profound antipolymer activity of ζ-substituted Hb S (Hb ζ2βS2) is less certain. X-ray crystallographic studies conducted at 1.95Å resolution revealed that liganded (CO-) Hb ζ2βS2 is trapped in a tense (T-state) quaternary structure, rather than in a relaxed (R-state) structure that is characteristic of nearly all liganded hemoglobins. Specifically, CO-Hb ζ2βS2 exhibited several intact T-state intersubunit salt-bridge/hydrogen-bond interactions, a preserved T-state ζ1-β2 (ζ2-β1) interface, and a characteristically enlarged T-state central water cavity and β-cleft. This structure wrongly predicts that liganded Hb ζ2βS2 will be included, rather than excluded, from the deoxyHb S polymer; and suggests that changes in the positions or the biochemical identities of individual amino acids, rather than the overall quaternary structure of liganded Hb ζ2βS2, are the chief determinants of its antipolymer activity. To define key differences in the structures of T-state deoxyHb α2βS2 and CO-Hb ζ2βS2, we superposed their corresponding globin subunits and calculated the specific displacement of individual amino-acid residues as root mean square deviation (rmsd) values. Among βS-chain residues, α→ζ exchange effects a significant 1.9Å shift in the position of the pathological βSVal6, and correspondingly large displacements of βThr4 (2.2Å) and βAsn19 (1.4Å); each repositioning predicts weakening of an intermolecular interaction that would otherwise stabilize the deoxyHb S polymer. Similar superposition analyses of the α and ζ chains reveal a significant displacement of αPro114 (1.3Å), a well-described determinant of deoxyHb S polymerization that is conserved between the two α-like subunits. Three additional α-chain residues that stabilize the deoxyHb S polymer undergo nonconservative replacement in the ζ-globin chain, but are not materially repositioned: αHis20→ζGln (basic→neutral polar), αAsn68→ζAsp (neutral polar→acidic), and αAsn78→ζGly (neutral polar→neutral). While all three replacements are predicted to weaken or ablate intermolecular contacts, the αHis20→ζGln substitution is particularly noteworthy as it reproduces the specific mutation that defines the naturally occurring anti-sickling variant αLe Lamentin. Finally, we considered the possibility that ζ-substituted Hb S is fully excluded from the deoxyHb S polymer--and therefore reduces the rate of its assembly--by comparing the crystal packing of the two hemoglobins. While deoxyHb α2βS2 packs in a familiar two-strand structure, CO-Hb ζ2βS2 assembles into a unique trimeric arrangement comprising three lateral heterotetramers, each of which interacts with an axial heterotetramer that is constituent to a separate trimer assembly. This remarkable structure is sustained by intermolecular interactions that are distinct from those observed for deoxyHb S. Moreover, the calculated buried solvent-accessible surface area for CO-Hb ζ2βS2 (4806Å2) is nearly two-fold higher than for deoxyHb α2βS2 (2510Å2), suggesting that Hb ζ2βS2 exists in solution as a stable trimer of heterotetramers, and validating the hypothesis that Hbs α2βS2 and ζ2βS2 do not co-assemble in solution. In sum, our crystal analyses indicate that the antipolymer activities of liganded Hb ζ2βS2 arise through movements in the positions of βS-chain residues, and through changes in the identities of α-chain residues. Our studies also demonstrate a novel packing structure for T-state liganded Hb ζ2βS2 that is consistent with its exclusion from the deoxyHb S polymer. These data account for the significant antipolymer activity of ζ-substituted Hb S, and recommend the utility of therapeutic approaches to SCD that are based upon α-globin subunit exchange. Disclosures Safo: Baxter and AesRx companies have licensed our patented antisickling compounds. Consulted with AesRx LLC during phase I clinical studies of the antisickling compound, 5HMF for the treatment of sickle cell disease: #7160910; #7119208 Patents & Royalties, Consultancy, Research Funding.

By using transgenic methodologies, we have produced a number of mouse/human chimeric hemoglobins containing adult mouse and human embryonic globin chains. A detailed analysis of the oxygen binding properties of these proteins identifies the dominant role played by the specific beta-type globin chains in the control of the oxygen binding characteristics. Further analysis traces the origins of these effects to alterations in the properties of the T states of these proteins. The human zeta/mouse beta chimeric protein has been crystallized, and its structure has been determined by X-ray diffraction to a resolution of 2.1 A with R (R(free)) values of 21.6% (24.9%). Close examination of the structure indicates that the subunit interfaces contain contacts which, although different from those present in either the parent human or the parent mouse proteins, retain the overall stabilizing interactions seen in other R state hemoglobins.

Abstract 1093 The high-level accumulation of β globin in mature erythrocytes requires a correspondingly high level of its encoding mRNA in terminally differentiating erythroid progenitors. We recently identified two RNA-binding proteins–AUF1 and YB1–that appear to regulate levels of β-globin mRNA in these cells by assembling a cytoplasm-restricted RNA-protein 'β-complex' on its 3'UTR. The function of the β-complex was predicted by in vitro analyses mapping it to a cis-acting determinant of β-globin mRNA stability, and subsequently validated by in vivo siRNA studies demonstrating that simultaneous knockdown of AUF1 and YB1 ablated the β-complex and coordinately reduced the accumulation of β-globin mRNA in K562 cells. Although both AUF1 and YB1 are ubiquitously expressed, studies in cultured cells and in Epo-induced CD34+ primary cells indicated that their β-globin mRNA-specific regulatory properties are restricted to erythroid cells during later stages of terminal differentiation. Based upon these observations, we reasoned that AUF1 and YB1 undergo erythroid and differentiation stage-restricted alterations that permit their assembly into the mRNA-regulatory β-complex. Our analyses of AUF1 focused on three structural isoforms, observed in K562 cells, resulting from alternative pre-mRNA processing events that retain or exclude exon 7. GST-AUF1 fusion isoforms that retain exon 7 fail to bind the β-globin 3'UTR in vitro, while related isoforms that exclude exon 7 bind the 3'UTR with high efficiency. These results, which implicate the importance of exon 7 exclusion to AUF1 function, were subsequently validated in intact K562 cells using an AUF1 isotype-specific siRNA knockdown strategy. In these in vivo experiments, a reduction in exon 7-excluded AUF1 effected a two-fold decrease in steady-state β-globin mRNA, while similar reductions in exon 7-retained AUF1 isoforms had no measurable effect. The isotype-specific mRNA-binding characteristics of AUF1 may be particularly important during terminal differentiation: Epo-induced CD34+ cells display an increase in exon 7-excluded AUF1, paralleling their capacity to assemble a regulatory β-complex in vitro. Among several possible mechanisms, we asked whether the isoform-specific function of AUF1 might relate to the unusually high number (20) of phosphorylation-capable residues encoded by exon 7. In vitro analyses were fully consistent with this possibility, demonstrating that the β-globin mRNA-binding activity of exon 7-retained AUF1 could be restored by prior dephosphorylation. These experiments suggest that post-transcriptional regulation of β-globin mRNA during erythroid differentiation is likely to be effected by alternative splicing of AUF1 pre-mRNA that eliminates phosphorylation-active exon 7 amino-acids in the translated protein. Based upon these results, we reasoned that related post-translational processes might similarly regulate the β-globin mRNA-binding specificity of YB1. Our analyses focused on a specific residue (Ser102) that is a known target for regulatory phosphorylation and can be experimentally identified using a Ser102 phospho-specific YB1 antibody. In in vitro studies with K562 cytoplasmic extract, which contains both phospho- and dephospho- forms of YB1, we observed that only dephospho-YB1 adheres to the β-globin 3'UTR; likewise, in in vivo studies of CD34+ cells we noted a substantial increase in the ratio of dephospho:phospho-YB1 following Epo induction. Both experiments indicate the likely importance of this post-translational process to the function of YB1 during terminal differentiation. Confirmatory studies are currently being conducted in vivo using an epitope-tagged YB1 containing a position 102 Ser->Ala substitution. Collectively, our analyses indicate that the β-globin mRNA-binding specificities of AUF1 and YB1–and, hence, their corresponding regulatory activities–are determined by post-transcriptional and -translational events. This work suggests mechanisms through which erythroid progenitors can maintain dynamic regulatory control during an interval when transcriptional processes are beginning to silence, and identifies new pathways that can be therapeutically targeted in patients with congenital disorders of β-globin gene expression. Disclosures: No relevant conflicts of interest to declare.

4059 Poster Board III-994 Transgenic approaches to β thalassemia and sickle cell disease require viral vectors that express high levels of therapeutic β-like globin proteins. We recently proposed that the overall expression of these transgenes would likely be improved by structural modifications that prolong the cytoplasmic half-lives of their encoded mRNAs. Relevant experiments from our laboratory have previously linked the constitutively high stability of β-globin mRNA to a region of its 3'UTR that appears to interact with at least two distinct cytoplasmic mRNA-stabilizing factors, and is predicted to form an imperfect stem-loop (SL) structure. Based upon these findings, we conducted enzymatic secondary-structure mapping studies of the β-globin 3'UTR, unequivocally validating the existence of the predicted functional stem-loop element. We subsequently reasoned that the constitutive half-life of β-globin mRNA might be prolonged by the insertion of multiple SL motifs into its 3'UTR, resulting in increased levels of the mRNA–and its encoded β-globin product–in terminally differentiating erythroid cells. To test this hypothesis, we constructed full-length β-globin genes containing either wild-type 3'UTRs, or variant 3'UTRs that had been modified to contain either two or three tandem SL motifs. Each gene was identically linked to a tetracycline-suppressible promoter, permitting pulse-chase mRNA stability analyses to be conducted in vivo in intact cultured cells. Erythroid-phenotype K562 cells were transiently transfected with SL-variant and control wild-type β-globin genes, exposed to tetracycline, and levels of β-globin mRNA determined by qRT-PCR at defined intervals using tet-indifferent β-actin mRNA as internal control. Relative to wild-type β-globin mRNA, SL-duplicate β-globin mRNAs displayed a position-dependent two-fold increase in cytoplasmic half-life; SL-triplicate β-globin mRNAs did not exhibit any additional stability. These experiments confirm the existence of a defined SL structure within the β-globin 3'UTR, and demonstrate that duplication of this motif can substantially increase the stability of β-globin mRNA. We subsequently designed a series of experiments to elucidate post-transcriptional processes involved in mRNA hyperstability. These studies required the construction of HeLa cells that stably express either wild-type β-globin mRNA (11 subclones) or SL-duplicate β-globin mRNAs (10 subclones). Preliminary analyses indicate an approximate 1.5-fold increase in the median steady-state expression of SL-duplicate genes, consistent with a prolongation in the half-life of its encoded mRNA. While formal mRNA stability studies are not yet complete, early data appear to replicate results from experiments conducted in transiently transfected cells. We have also initiated structural studies to link differences in the stability of SL-variant β-globin mRNA to alterations in its poly(A) tail. Using an RNase H-based strategy, we identified a previously unknown poly(A)-site heterogeneity–of undetermined significance–affecting both wild-type and SL-duplicate β-globin mRNAs. Finally, we recently isolated fifty-four K562 subclones expressing SL-duplicate or control β-globin mRNAs; parallel analyses of these cells will permit the cell-specificity of β-globin SL-directed mRNA stabilization to be investigated in detail. Results from each of these studies will be immediately applicable to the design of high-efficiency therapeutic transgenes for β thalassemia and sickle-cell disease. Disclosures: No relevant conflicts of interest to declare.

The normal expression of human β globin is critically dependent upon the high stability of its encoding mRNA. The mechanism that protects β-globin mRNA from premature degradation--including the positions of cis-acting stability determinants, the identities of relevant trans-acting factors, and the processes through which they interact--are poorly understood. We have designed and executed a series of experiments that detail the critical importance of the 3′UTR to the high constitutive stability of β-globin mRNA. To identify mRNA-stability determinants in this region, we constructed a wild-type β-globin gene (βWT), as well as 17 derivative genes containing site-specific 3′UTR hexanucleotide substitutions (βMUT1-βMUT17), each under the transcriptional control of a tetracycline-response element (TRE). In cultured cells that express a corresponding transcriptional transactivator, the expression of TRE-linked βWT and βMUT genes can be rapidly silenced by adding tetracycline to the culture medium, permitting the stabilities of the cognate mRNAs to be established using a transcriptional chase approach. Two of the 17 mRNAs, carrying adjacent hexanucleotide substitutions (βMUT12 and βMUT13), were destabilized in intact HeLa cells, identifying a sequence that is critical to β-globin mRNA stability. Three potentially important trans-acting factors that bind to this region were subsequently isolated using an in vitro affinity-enrichment method. One of the proteins was unequivocally identified by mass spec analysis to be nucleolin, a ubiquitous nuclear-cytoplasmic factor that exhibits RNA helicase activity and is reported to stabilize several non-erythroid mRNAs. A link between this factor and β-globin mRNA stability was provided by in vitro studies demonstrating that purified nucleolin binds tightly to the βWT 3′UTR but poorly to both βMUT12 and βMUT13 3′UTRs. This result was validated by RNA-immunoprecipitation (RIP) analyses confirming a strong interaction between nucleolin and βWT mRNA in intact cells that is fully ablated by MUT12 or MUT13 hexanucleotide substitutions. The critical importance of nucleolin binding to the stability of β-globin mRNA may relate to a stem-and-loop motif within its 3′UTR that is predicted by mRNA-folding algorithms. This structure contains the nucleolin-binding site on its right half-stem, opposite a putative binding site for αCP, a 34 kDa factor that stabilizes α-globin mRNA, on the left half-stem. Surprisingly, recombinant αCP displays a low affinity for the full-length β-globin 3′UTR, while binding avidly to the isolated left half-stem as well as to full-length β-globin 3′UTRs that contain stem-disrupting mutations. These results indicate that high-order structures within the β-globin 3′UTR, if permitted to form, may interfere with αCP function in vivo. Based upon our studies, we suggest that nucleolin binding is required to relax a highly stable stem-and-loop motif within the β-globin 3′UTR, exposing a functional binding site for the mRNA-stabilizing factor αCP. Thus, we identify a cis-element and a specific trans-acting factor that participate in stabilizing β-globin mRNA, and suggest a mechanism through which they are likely to act in vivo. The full elucidation of this process will clearly benefit the design of therapeutic transgenes for individuals with β-globin gene defects, and may additionally facilitate the conception of novel therapies intended to differentially regulate the stabilities of βS- and γ-globin mRNAs in individuals with sickle cell disease.