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The normal expression of β-globin protein in mature erythrocytes is critically dependent on post-transcriptional events in erythroid progenitors that ensure the high stability of β-globin mRNA. Previous work has revealed that these regulatory processes require AUF-1 and YB-1, two RNA-binding proteins that assemble an mRNP β-complex on the β-globin 3'UTR. Here, we demonstrate that the β-complex organizes during the erythropoietic interval when both β-globin mRNA and protein accumulate rapidly, implicating the importance of this regulatory mRNP to normal erythroid differentiation. Subsequent functional analyses link β-complex assembly to the half-life of β-globin mRNA in vivo, providing a mechanistic basis for this regulatory activity. AUF-1 and YB-1 appear to serve a redundant post-transcriptional function, as both β-complex assembly and β-globin mRNA levels are reduced by coordinate depletion of the two factors, and can be restored by independent rescue with either factor alone. Additional studies demonstrate that the β-complex assembles more efficiently on polyadenylated transcripts, implicating a model in which the β-complex enhances the binding of PABPC1 to the poly(A) tail, inhibiting mRNA deadenylation and consequently effecting the high half-life of β-globin transcripts in erythroid progenitors. These data specify a post-transcriptional mechanism through which AUF1 and YB1 contribute to the normal development of erythropoietic cells, as well as to non-hematopoietic tissues in which AUF1- and YB1-based regulatory mRNPs have been observed to assemble on heterologous mRNAs.

Erythropoietic protoporphyria (EPP) is a rare inherited disorder of the heme biosynthesis pathway resulting in the accumulation of protoporphyrins in the blood, erythrocytes, and other tissues. Because of a gene mutation in the FECH gene, ferrochelatase, the enzyme involved in the final step of heme synthesis, is deficient in these patients. Although the major symptom of this disorder is photosensitivity, rarely, it can cause progressive liver disease requiring liver transplantation (LT). However, LT is not curative and only bone marrow transplantation (BMT) can correct the underlying enzymatic defect. Because liver disease results from accumulation of protoporphyrin in the liver, LT without hematopoietic stem cell transplantation leaves the new liver at risk for similar EPP-related damage. A handful of pediatric patients undergoing sequential LT and stem cell transplantation have been described in the literature; however, to date none has been described in detail in adults. We report a case of an adult male with EPP and liver failure who successfully underwent a sequential liver and hematopoietic stem cell transplantation (HSCT).

The normal accumulation of β-globin protein in terminally differentiating erythroid cells is critically dependent on the high stability of its encoding mRNA. The molecular basis for this property, though, is incompletely understood. Factors that regulate β-globin mRNA within the nucleus of early erythroid progenitors are unlikely to account for the constitutively high half-life of β-globin mRNA in the cytoplasm of their anucleate erythroid progeny. We conducted in vitro protein-RNA binding analyses that identified a cytoplasm-restricted β-globin messenger ribonucleoprotein (mRNP) complex in both cultured K562 cells and erythroid-differentiated human CD34+ cells. This novel mRNP targets a specific guanine-rich pentanucleotide in a region of the β-globin 3′untranslated region that has recently been implicated as a determinant of β-globin mRNA stability. Subsequent affinity-enrichment analyses identified AUF-1 and YB-1, 2 cytoplasmic proteins with well-established roles in RNA biology, as trans-acting components of the mRNP. Factor-depletion studies conducted in vivo demonstrated the importance of the mRNP to normal steady-state levels of β-globin mRNA in erythroid precursors. These data define a previously unrecognized mechanism for the posttranscriptional regulation of β-globin mRNA during normal erythropoiesis, providing new therapeutic targets for disorders of β-globin gene expression.

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.

Different types of human hemoglobins (Hbs) consisting of various combinations of the embryonic, fetal, and adult Hb subunits are present at certain times during development representing a major paradigm of developmental biology that is still not understood and one which we address here. We show that the subunit interfaces of these Hbs have increasing bonding strengths as demonstrated by their distinct distribution of tetramers, dimers, and monomers during gel filtration at very low-Hb concentration. This maturation is mediated by competition between subunits for more favorable partners with stronger subunit interactions. Thus, the protein products of gene expression can themselves have a role in the developmental process due to their intrinsic properties.

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.

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.

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.

Alternative pre-mRNA splicing is a prevalent mechanism in mammals that promotes proteomic diversity, including expression of cell-type specific protein isoforms. We characterized a role for RBM38 (RNPC1) in regulation of alternative splicing during late erythroid differentiation. We used an Affymetrix human exon junction (HJAY) splicing microarray to identify a panel of RBM38-regulated alternatively spliced transcripts. Using microarray databases, we noted high RBM38 expression levels in CD71(+) erythroid cells and thus chose to examine RBM38 expression during erythroid differentiation of human hematopoietic stem cells, detecting enhanced RBM38 expression during late erythroid differentiation. In differentiated erythroid cells, we validated a subset of RBM38-regulated splicing events and determined that RBM38 regulates activation of Protein 4.1R (EPB41) exon 16 during late erythroid differentiation. Using Epb41 minigenes, Rbm38 was found to be a robust activator of exon 16 splicing. To further address the mechanism of RBM38-regulated alternative splicing, a novel mammalian protein expression system, followed by SELEX-Seq, was used to identify a GU-rich RBM38 binding motif. Lastly, using a tethering assay, we determined that RBM38 can directly activate splicing when recruited to a downstream intron. Together, our data support the role of RBM38 in regulating alternative splicing during erythroid differentiation.

A variant Hb ζ2β2sthat is formed from sickle hemoglobin (Hb S; α2β2s) by exchanging adult α-globin with embryonic ζ-globin subunits shows promise as a therapeutic agent for sickle-cell disease (SCD). Hb ζ2β2sinhibits the polymerization of deoxygenated Hb Sin vitroand reverses characteristic features of SCDin vivoin mouse models of the disorder. When compared with either Hb S or with normal human adult Hb A (α2β2), Hb ζ2β2sexhibits atypical properties that include a high oxygen affinity, reduced cooperativity, a weak Bohr effect and blunted 2,3-diphosphoglycerate allostery. Here, the 1.95 Å resolution crystal structure of human Hb ζ2β2sthat was expressed in complex transgenic knockout mice and purified from their erythrocytes is presented. When fully liganded with carbon monoxide, Hb ζ2β2sdisplays a central water cavity, a ζ1–βs2 (or ζ2–βs1) interface, intersubunit salt-bridge/hydrogen-bond interactions, C-terminal βHis146 salt-bridge interactions, and a β-cleft, that are highly unusual for a relaxed hemoglobin structure and are more typical of a tense conformation. These quaternary tense-like features contrast with the tertiary relaxed-like conformations of the ζ1βs1 dimer and the CD and FG corners, as well as the overall structures of the heme cavities. This crystallographic study provides insights into the altered oxygen-transport properties of Hb ζ2β2sand, moreover, decouples tertiary- and quaternary-structural events that are critical to Hb ligand binding and allosteric function.

Severe coagulant factor VII (FVII) deficiency in postpubertal dizygotic twin males results from two point mutations in the FVII gene, a promoter region T→C transition at -60 and a His-to-Arg substitution at amino acid 348; both mutations prevent persistence of plasma functional FVII. This report documents longitudinal laboratory measurements from infancy to adulthood of FVII coagulant activity (FVII:C) in the twin FVII-deficient patients; it also details specific biochemical analyses of the -60 T→C mutation. The results revealed FVII:C levels of less than 1% in infancy that remain severely decreased through puberty and into adulthood. In-vitro analyses utilizing hepatocyte nuclear factor 4α (HNF4α) co-transfection and a chromatin immunoprecipitation assay indicate that the -60 T→C mutation severely diminishes functional interaction between the FVII promoter and transcription factor HNF4α. The importance of interaction between the FVII gene and HNF4α in normal FVII expression provides an in-vivo illustration of the regulated expression of an autosomal gene encoding a coagulation protein. The constancy of FVII:C and peripubertal patient symptomatology reported here illustrates androgen-independent expression in contrast to expression with an analogous mutation in the promoter region of the gene encoding coagulation FIX.

The distal 3'UTR of prothrombin mRNA exhibits significant sequence heterogeneity reflecting an inexact 3'-cleavage/polyadenylation reaction. This same region encompasses a single-nucleotide polymorphism that enhances the normal post-transcriptional processing of nascent prothrombin transcripts. Both observations indicate the importance of 3'UTR structures to physiologically relevant properties of prothrombin mRNA. Using a HepG2-based model system, we mapped both the primary structures of reporter mRNAs containing the prothrombin 3'UTR, as well as the secondary structures of common, informative 3'UTR processing variants. A chromatographic method was subsequently employed to assess the effects of structural heterogeneities on the binding of candidate trans-acting regulatory factors. We observed that prothrombin 3'UTRs are constitutively polyadenylated at seven or more positions, and can fold into at least two distinct stem-loop conformations. These alternate structures expose/sequester a consensus binding site for hnRNP-I/PTB-1, a trans-acting factor with post-transcriptional regulatory properties. hnRNP-I/PTB-1 exhibits different affinities for the alternate 3'UTR secondary structures in vitro, predicting a corresponding regulatory role in vivo. These analyses demonstrate a critical link between the structure of the prothrombin 3'UTR and its normal function, providing a basis for further investigations into the molecular pathophysiology of naturally occurring polymorphisms within this region.

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.

The normal expression of human beta globin is critically dependent upon the constitutively high stability of its encoding mRNA. Unlike with alpha-globin mRNA, the specific cis-acting determinants and trans-acting factors that participate in stabilizing beta-globin mRNA are poorly described. The current work uses a linker-scanning strategy to identify a previously unknown determinant of mRNA stability within the beta-globin 3' untranslated region (3'UTR). The new determinant is positioned on an mRNA half-stem opposite a pyrimidine-rich sequence targeted by alphaCP/hnRNP-E, a factor that plays a critical role in stabilizing human alpha-globin mRNA. Mutations within the new determinant destabilize beta-globin mRNA in intact cells while also ablating its 3'UTR-specific interaction with the polyfunctional RNA-binding factor nucleolin. We speculate that 3'UTR-bound nucleolin enhances mRNA stability by optimizing alphaCP access to its functional binding site. This model is favored by in vitro evidence that alphaCP binding is enhanced both by cis-acting stem-destabilizing mutations and by the trans-acting effects of supplemental nucleolin. These studies suggest a mechanism for beta-globin mRNA stability that is related to, but distinct from, the mechanism that stabilizes human alpha-globin mRNA.

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.

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.

The activated form of prothrombin plays pivotal roles in the regulation of crucial coagulation, fibrinolytic, and cellular processes. Among several congenital genetic defects affecting the prothrombin gene, a G→A mutation at position 20210—the accepted polyadenylation site—has been linked to hyperprothrombinemia and a corresponding increase in venous and arterial thrombotic risk. The current study substantiates the hypothesis that the 20210A mutation effects posttranscriptional dysregulation of the prothrombin messenger RNA (mRNA). Moreover, data from experiments carried out in fresh liver tissue indicate that the 20210A mutation does not affect prothrombin mRNA stability but, rather, effects a change in the location of the 3′-cleavage/polyadenylation reaction. Based upon this evidence, we propose an alternate model for the dysregulated expression of the prothrombin 20210A gene that does not require a change in the stability of its mRNA.

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.

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.

The human alpha-globin gene cluster contains three functional genes zeta, alpha 2 and alpha 1. The zeta-globin gene is expressed exclusively in the primitive erythroblasts of the embryonic yolk sac and is selectively silenced during the transition from primitive to definitive erythropoesis. The two alpha-globin genes are expressed through development; they are expressed at equivalent levels in embryonic cells at a 2.6:1 ratio of alpha 2:alpha 1 in fetal and adult cells. The dominant contribution of the alpha 2-globin locus to overall expression of adult alpha-globin is reflected in the more severe phenotype resulting from mutations that affect this locus. Developmental silencing of the zeta-globin gene reflects both transcriptional and posttranscriptional mechanisms. Transcriptional silencing is mediated by an interaction between the zeta-globin gene promoter and a silencer located in the 3' flanking region. This transcriptional silencing is only partial, and residual levels of zeta-globin mRNA are subject to subsequent degredation. This instability of zeta-globin mRNA relative to that of alpha-globin mRNA reflects differences in their respective 3'UTR segments; the zeta-globin mRNA 3'UTR has a lower affinity for a sequence-specific mRNP stability complex which assembles at this site. The alpha-globin mRNA assembles this complex at a higher efficiency and mutations which interfere with 3'UTR function result in corresponding loss of alpha-globin gene expression. These data outline a developmental pathway for the alpha-globin gene cluster which reflects transcriptional and posttranscriptional controls.

Standard methods for assessing mRNA stabilities in intact cells are labor-intensive and can generate half-life (t(1/2)) measures that are both imprecise and inaccurate. We describe modifications to a conventional tetracycline-conditional transcriptional chase method for analyzing mRNA stability that significantly simplify its conduct, while generating highly reproducible and accurate t(1/2) values. The revised method-which is conducted as a reverse time course, and which accounts for interval expansion in the number of cultured cells-is validated for the analyses of mRNAs with both short and long half-lives. This approach facilitates accurate assessment of mRNA metabolism, providing a user-friendly tool for detailed investigations into their structures and functions, as well as the processes that contribute to their post-transcriptional regulation.