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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.

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.

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.

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.

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.

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.

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.

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 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.

Post-transcriptional events that regulate the stabilities of one or more specific mRNAs are increasingly recognized for their importance to cell development and differentiation: genome-wide analyses attribute ∼50% of changes in gene expression to alterations in the stabilities of their encoded mRNAs. Processes that differentially regulate the half-lives of mRNAs are particularly important in definitive erythropoiesis, as they control the relative levels of actively translating transcripts in the interval when the nucleus is transcriptionally silenced and is ultimately extruded. We recently identified AUF-1 (AU-binding factor 1; hnRNP D) as a trans-acting factor that stabilizes human β-globin mRNA, and noted previous reports that this RNA-binding protein also regulates the half-lives of other mRNAs encoding factors that are critical to normal erythropoiesis, including p16INK4a (CDKN2A), p21 (CDKN1A), cyclin D1 (CCND1), and thymidylate synthase. Based upon this evidence, we posited that AUF-1 plays a role in post-transcriptional erythropoiesis that is far broader than previously imagined. Our analyses of umbilical cord blood CD34+ cells validated this expectation, demonstrating the presence of two AUF-1 isoforms (p45AUF1 and p40AUF1) that differ by the inclusion/exclusion of exon 7-encoded sequence. Cells that are induced to erythroid differentiation initially express p45AUF1 and p40AUF1 at equal levels, but shift after several days to express only the p40AUF1 isoform. In contrast, cells that are induced to granulocyte-monocyte differentiation initially express only p45AUF1, and continue to express this single isoform through the remainder of development. These observations demonstrate lineage-specific AUF-1 isoform expression that is likely to be important to both erythroid and non-erythroid developmental programs. We subsequently investigated the functional consequences of the erythropoietic AUF-1 isoform switch by analyzing the abilities of p45AUF1 and p40AUF1 to stabilize informative erythroid-specific and -restricted mRNAs. Using an AUF-1 isoform-specific siRNA knockdown strategy, we demonstrated that a reduction in p40AUF-1 (but not p45AUF-1) effected a two-fold decrease in β-globin mRNA in K562 cells that we engineered to express this gene at high levels. p45AUF1 and p40AUF1 knockdown had a different effect on mRNAs encoding cyclin D1 and p21, both of which were unaffected by single-isoform depletion but--in contrast to β-globin mRNA--were increased by coordinate knockdown of both p45AUF1 and p40AUF1. While we expected that the erythropoietic AUF-1 isoform switch was likely due to alternative splicing of exon 7 from the AUF-1 pre-mRNA, we observed instead that the switch results from the transfer of p40AUF1 from the nucleus to the cytoplasm during the developmental interval that immediately precedes nuclear extrusion. This observation implicates developmentally regulated trafficking of p40AUF1 during erythropoiesis that is consistent with its demonstrated post-transcriptional mRNA-stabilizing effects. Finally, we assessed expression of HuR--a competitive antagonist of AUF-1 that has been implicated in stabilizing the mRNAs encoding FasL and cyclin E1 (CCNE1)--in erythroid-induced CD34+ cells, and observed that levels of HuR mRNA increase significantly during the developmental interval that coincides with the erythropoietic p45:p40AUF1 isoform switch. Collectively, the lineage- and developmental-stage specific expression of p45AUF1, p40AUF1, and HuR--combined with their mRNA-specific binding properties--illustrate both the existence and the complexity of post-transcriptional regulatory programs that contribute to the normal development of both erythroid and nonerythroid cells. We are currently conducting RNA-seq analyses in erythroid-differentiated CD34+ cells to identify transcripts that differentially bind p45AUF1, p40AUF1, and HuR; as well as factor-specific shRNA knock-down analyses to characterize corresponding functional effects. In sum, our present work describes a novel mechanism through which erythroid progenitors maintain dynamic regulatory control during an interval when transcriptional processes are silenced. Disclosures: No relevant conflicts of interest to declare.

Abstract 1020 The normal expression of Hb A in humans requires the high-level stability of α - and β-globin mRNAs in both transcriptionally active and transcriptionally silenced erythroid progenitors. In contrast to α -globin–whose stability is known to be enhanced by an mRNA-protein (mRNP) complex that assembles on a specific pyrimidine-rich track within its 3'UTR–the structure(s) and mechanism(s) that underlie the high stability of human β-globin mRNA remain poorly defined. We recently identified two RNA-binding proteins, AUF-1 and YB-1, that regulate levels of β-globin mRNA in erythroid progenitors by assembling a cytoplasm-restricted mRNP 'β-complex' on its 3'UTR. The function of the β-complex was predicted by in vitro analyses that mapped its binding to a cis-acting determinant of β-globin mRNA stability, and by in vivo siRNA studies demonstrating that simultaneous knockdown of AUF-1 and YB-1 coordinately ablated the β-complex and coordinately reduced the accumulation of β-globin mRNA in K562 cells. The biological importance of the β-complex was subsequently confirmed in human hematopoietic stem cells, where shRNA-mediated knock-down of AUF-1 or YB-1 effected lower levels of β-globin mRNA in cells induced to the erythroid lineage, again implicating their participation in post-transcriptional mechanism(s) regulating the stability of β-globin mRNA. To unambiguously link β-complex activity to β-globin mRNA half-life, we conducted formal in vivo mRNA stability analyses in K562 cells using a β-globin mRNA-specific tetracycline-conditional transcriptional chase strategy. A derivative β-globin mRNA carrying a 5-nt substitution that totally disrupts β-complex assembly (βMut mRNA) displayed a lower half-life than wild-type β-globin mRNA (βWT mRNA), confirming the participation of the β-complex in post-transcriptional regulatory processes. Parallel poly(A) tail length analyses indicated a possible mechanism for this activity, revealing that the βMut mRNA had a shortened steady-state poly(A) tail that truncated faster than the poly(A) tail on βWT mRNA, suggesting a functional interaction between the β-complex and poly(A) tail-associated factors. This observation is fully consistent with the known importance of deadenylation to processes regulating the decay of heterologous mRNAs in several other experimental systems. Subsequent studies supported our model for β-complex/poly(A) tail interaction: electrophoretic gel mobility-shift analyses demonstrated that the β-complex readily assembles on polyadenylated β-globin 3'UTRs but not on corresponding deadenylated 3'UTRs, while RNA affinity capture experiments using K562 cytoplasmic extracts demonstrated that a polyadenylated βWT 3'UTR retains poly(A) binding protein (PABP), while a similar β-complex-deficient βMut 3'UTR fails to bind PABP. Ongoing co-immunoprecipation studies are expected to determine whether the β-complex and PABP are tethered by an interval of mRNA or, alternately, interact directly via a protein-protein interaction. Based upon our previous structural and functional analyses indicating that AUF-1 and YB-1 act redundantly to regulate the cytoplasmic level of β-globin mRNA, we are currently investigating the hypothesis that these two factors also display redundant interactions with the poly(A) tail and its trans-acting binding factors. Our initial RNA affinity analyses confirm this expectation, demonstrating that K562 extracts depleted of either AUF-1 or YB-1 (using an shRNA-knock-down strategy) both maintained the ability to assemble a β-complex as well as facilitate PABP binding to a the polyadenylated βWT 3'UTR. We are presently testing AUF-1 and YB-1 for corresponding functional redundancy (i.e., their abilities to independently induce βWT mRNA stability) using in vivo mRNA tethering experiments in which AUF-1 or YB-1 can be structurally modified to promote their independent interaction with the β-complex binding site. Altogether, these experiments demonstrate that the β-complex, through its component mRNA-binding factors AUF-1 and YB-1, effects the high stability of β-globin mRNA by interacting with PABP. A detailed structural and mechanistic description of this process will be invaluable to the design of novel therapeutics for patients with congenital disorders of β-globin gene expression. Disclosures: No relevant conflicts of interest to declare.

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.

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.

Abstract 644 The efficient accumulation of hemoglobin in mature erythrocytes is critically dependent upon the high stabilities of mRNAs encoding human α- and β-globin proteins. These mRNAs are likely to be stabilized by interactions between one or more trans-acting regulatory factors that target defined cis-acting elements within their 3′UTRs. Several ubiquitous factors that are known to bind to the β-globin 3′UTR (including αCP, PTBP1, and nucleolin) are largely restricted to the nucleus and therefore unlikely to contribute to regulatory processes affecting β-globin mRNA in the cytoplasm. Consequently, we conducted a series of experiments that identify and characterize mRNA-binding factors that dictate the properties of β-globin mRNA in the cytoplasm of erythroid progenitor cells. Using electrophoretic gel mobility shift analyses (EMSA), we defined a characteristic mRNP complex that assembles on the β-globin 3′UTR in cytoplasmic extract–but not nuclear extract–prepared from erythroid K562 cells. This mRNP ‘β-complex’ appears to be erythroid-specific, as it fails to assemble in extracts prepared from non-erythroid HeLa or HEK cells. The 3′UTR binding site for the β-complex was identified using an EMSA-competition approach; remarkably, the target sequence is encompassed within a 12-nt region previously identified as a functional determinant of β-globin mRNA stability in in vivo analyses. Additional experiments fine-mapped the β-complex binding site to a GGGGG pentanucleotide motif within the mRNA-stabilizing region. The functional importance of the pentanucleotide was illustrated by mRNA decay experiments in intact erythroid K562 cells showing that full-length β-globin mRNAs are destabilized by introduction of the same GGGGG->CCGGG mutation that ablates β-complex assembly in EMSA analyses. To identify trans-factors that comprise the β-complex, we performed affinity chromatography using ssDNA probes corresponding to the β-complex binding motif. The native 3′UTR probe retained 42- and 47-kDa proteins, while a probe carrying the CCGGG mutation failed to bind either factor. Subsequent LC/MS/MS analyses identified the two proteins as YB-1 and AUF-1. The identities of these two mRNA-binding factors, which have previously been implicated in the post-transcriptional regulation of heterologous mRNAs, were subsequently confirmed by immunoblot of the protein-DNA complexes. Subsequent analyses suggested a functional role for both factors: EMSA supershift experiments confirmed that YB-1 is a component of the β-complex, and RNA immunoprecipitation analyses demonstrated that both YB-1 and AUF-1 specifically bind to β-globin mRNA in vivo in intact erythroid K562 cells. Collectively, these data identify two novel trans-acting factors that bind to cytoplasmic β-globin mRNA in an erythroid-specific fashion, at a site that dictates its stability in intact cells. We are currently engaged in siRNA knock-down experiments to validate experiments that suggest the importance of these trans-acting factors to the constitutive cytoplasmic stability of β-globin mRNA, as well as structural analyses intended to define RNA-protein and protein-protein interactions that are critical to normal functioning of the β-complex. The results of these experiments have obvious implications for the design of novel therapies for patients with congenital disorders of β-globin gene expression, including sickle cell disease and β thalassemia. 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 activated form of prothrombin (F.II) plays a pivotal role in physiologically important thrombotic and fibrinolytic processes. Heterozygotes for a G->A transversion at position 20210 of the F.II 3′UTR display elevated prothrombin levels and exhibit a corresponding increase in the risk of cardiac and cerebral thromboses. Previous studies of endogenous F.IIG and F.IIA mRNAs in primary liver cells, and analyses of transiently expressed F.IIG and F.IIA reporter mRNAs expressed in cultured heterologous cells, have reached different conclusions on the molecular mechanism through which the G20210A mutation effects F.II overexpression. To resolve this issue, we designed a cell-homologous culture system for directly assessing functional attributes of F.II reporter mRNAs, including their stabilities as well as their translational efficiencies. An hepatocellular-phenotype HepG2 cell line was generated that stably expresses the tetracycline transactivator (tTA), a chimeric factor that mediates tetracycline-conditional silencing of genes linked to a tetracycline regulatory element (TRE). These cells, which permit the stabilities of mRNAs encoded by TRE-linked genes to be determined by transcriptional chase analysis, were validated in studies utilizing stable and unstable control mRNAs. TRE-linked β-globin reporter genes were subsequently constructed to contain the F.II 5′UTR as well as the full-length F.II 3′UTR and contiguous flanking region, with either the native G (βF.IIG) or the mutant A (βF.IIA) at position 20210. The βF.IIG and βF.IIA mRNAs displayed equal stabilities when transiently expressed in intact tTA-expressing HepG2 cells, an observation corroborated by formal studies in multiple cell lines containing stably-integrated TRE-linked βF.IIG and βF.IIA genes. This data suggests that the G20210A mutation is likely to effect clinical hyperprothrombinemia through inductive effects on the translational efficiency of the F.II mRNA rather than by altering its stability. The mechanistic basis for this property may reflect differences in the 3′-cleavage patterns exhibited by nascent F.IIG mRNA (occurring at nts 20210 and 20212) and F.IIA mRNA (occurring only at nt 20210). Consistent with this possibility, we observed that 20212-terminal F.IIG 3′UTRs fail to bind three cytoplasmic factors that exhibit a strong affinity for both 20210-terminal F.IIG and F.IIA 3′UTRs in vitro. A candidate binding site for one factor--at positions 20202-06--may be sterically inhibited by potential secondary structure that forms in 20212-, but not the 20210- terminal F.II mRNA 3′-cleavage isotypes. We propose that the differential affinity of this factor for 20210- and 20212-terminal F.II mRNAs may mediate a difference in the aggregate translational efficiencies of F.IIG and F.IIA mRNAs. To test this hypothesis, we established a sucrose-gradient fractionation method that estimates the translational efficiency of an individual mRNA by assessing the number of ribosomes that it carries. Initial studies indicate a difference in the ribosomal loading of βF.IIG and βF.IIA mRNAs that is consistent with their anticipated relative translational efficiencies. The results of these studies will provide a rational basis for the design of strategies to therapeutically manipulate the expression of F.II, and will afford potential insights into the regulation of other, evolutionarily related coagulation factors whose expression levels may define independent thrombotic risk.

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.