J. Eric Russell

Structural and Functional Analysis of an mRNP Complex That Mediates the High Stability of Human β-Globin mRNA

ABSTRACT Human globins are encoded by mRNAs exhibiting high stabilities in transcriptionally silenced erythrocyte progenitors. Unlike α-globin mRNA, whose stability is enhanced by assembly of a specific messenger RNP (mRNP) α complex on its 3′ untranslated region (UTR), neither the structure(s) nor the mechanism(s) that effects the high-level stability of human β-globin mRNA has been identified. The present work describes an mRNP complex assembling on the 3′ UTR of the β-globin mRNA that exhibits many of the properties of the stability-enhancing α complex. The β-globin mRNP complex is shown to contain one or more factors homologous to αCP, a 39-kDa RNA-binding protein that is integral to α-complex assembly. Sequence analysis implicates a specific 14-nucleotide pyrimidine-rich track within its 3′ UTR as the site of β-globin mRNP assembly. The importance of this track to mRNA stability is subsequently verified in vivo using mice expressing human β-globin transgenes that contain informative mutations in this region. In combination, the in vitro and in vivo analyses indicate that the high stabilities of the α- and β-globin mRNAs are maintained through related mRNP complexes that may share a common regulatory pathway.

Sequence Divergence in the 3′ Untranslated Regions of Human ζ- and α-Globin mRNAs Mediates a Difference in Their Stabilities and Contributes to Efficient α-to-ζ Gene Developmental Switching

ABSTRACT The developmental stage-specific expression of human globin proteins is characterized by a switch from the coexpression of ζ- and α-globin in the embryonic yolk sac to exclusive expression of α-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 ζ-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 ζ-globin mRNA. Using a transgenic mouse model system, we demonstrate that human ζ-globin mRNA is unstable in adult erythroid cells relative to the highly stable human α-globin mRNA. A critical determinant of the difference between α- and ζ-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 α- and ζ-globin 3′UTRs assemble a previously described mRNP stability-determining complex, the α-complex, with distinctly different affinities. The diminished efficiency of α-complex assembly on the ζ 3′UTR results from a single C→G nucleotide substitution in a crucial polypyrimidine tract contained by both the human α- and ζ-globin mRNA 3′UTRs. A potential pathway for accelerated ζ-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 ζ-globin gene silencing in fetal and adult erythroid cells in which posttranscriptional controls play a central role by providing for accelerated clearance of ζ-globin transcripts.

A 3′UTR mutation affects β‐globin expression without altering the stability of its fully processed mRNA

Summary. 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 β thalassaemia who carry a β‐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 β‐globin mRNA stability. We studied the impact of the Δ13 mutation on β‐globin gene expression in vitro and in vivo. The adverse effect of the Δ13 mutation on β‐globin expression was confirmed in studies utilizing reticulocytes from a βΔ13 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 βΔ13 mRNA to assemble an mRNA‐stabilizing mRNP ‘β‐complex’. Unexpectedly, functional tests in both primary erythroid cells and in a transgenic mouse model demonstrated that the βΔ13 mRNA was fully stable, suggesting that the Δ13 mutation affects accumulation of the fully processed mRNA at an earlier step. Consistent with this, there was a relative excess of unprocessed βΔ13 mRNA in erythroid progenitors from a βΔ13 heterozygote. Taken together, these results define a new thalassaemic determinant, which acts to decrease β‐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 β‐globin gene expression.

The RNA Binding Protein RBM38 (RNPC1) Regulates Splicing during Late Erythroid Differentiation

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.

Human embryonic, fetal, and adult hemoglobins have different subunit interface strengths. Correlation with lifespan in the red cell

The different types of naturally occurring, normal human hemoglobins vary in their tetramer–dimer subunit interface strengths (stabilities) by three orders of magnitude in the liganded (CO or oxy) state. The presence of embryonic ζ‐subunits leads to an average 20‐fold weakening of tetramer–dimer interfaces compared to corresponding hemoglobins containing adult α‐subunits. The dimer–monomer interfaces of these hemoglobins differ by at least 500‐fold in their strengths; such interfaces are weak if they contain ζ‐subunits and exchange with added β‐subunits in the form of β4 (HbH) significantly faster than do those with α‐subunits. Subunit exchange occurs at the level of the dimer, although tetramer formation reciprocally influences the amount of dimer available for exchange. Competition between subunit types occurs so that pairs of weak embryonic hemoglobins can exchange subunits to form the stronger fetal and adult hemoglobins. The dimer strengths increase in the order Hb Portland‐2 (ζ2β2) < Hb Portland‐1 (ζ2γ2) ≅ Hb Gower‐1 (ζ2ε2) < Hb Gower‐2 (α2ε2) < HbF1 < HbF (α2γ2) < HbA2 (α2δ2), i.e., from embryonic to fetal to adult types, representing maturation from weaker to stronger monomer–monomer subunit contacts. This increasing order recapitulates the developmental order in which globins are expressed (embryonic → fetal → adult), suggesting that the intrinsic binding properties of the subunits themselves regarding the strengths of interfaces they form with competing subunits play an important role in the dynamics of protein assemblies and networks.

Expression and Developmental Control of the Human α‐Globin Gene Cluster

Abstract: The human α‐globin gene cluster contains three functional genes ζ, α2 and α1. The ζ‐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 α‐globin genes are expressed through development; they are expressed at equivalent levels in embryonic cells at a 2.6: 1 ratio of α2 : α1 in fetal and adult cells. The dominant contribution of the α2‐globin locus to overall expression of adult α‐globin is reflected in the more severe phenotype resulting from mutations that affect this locus. Developmental silencing of the ζ‐globin gene reflects both transcriptional and posttranscriptional mechanisms. Transcriptional silencing is mediated by an interaction between the ζ‐globin gene promoter and a silencer located in the 3′ flanking region. This transcriptional silencing is only partial, and residual levels of ζ‐globin mRNA are subject to subsequent degredation. This instability of ζ‐globin mRNA relative to that of α‐globin mRNA reflects differences in their respective 3′UTR segments; the ζ‐globin mRNA 3′UTR has a lower affinity for a sequence‐specific mRNP stability complex which assembles at this site. The α‐globin mRNA assembles this complex at a higher efficiency and mutations which interfere with 3′UTR function result in corresponding loss of α‐globin gene expression. These data outline a developmental pathway for the α‐globin gene cluster which reflects transcriptional and posttranscriptional controls.

Antisickling effects of an endogenous human alpha-like globin.

Gene replacement or gene reactivation therapies for sickle-cell disease (SCD) typically target the mutant βS-globin subunits of hemoglobin-S (α2βS2) for substitution by nonpathological β-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 α-globin subunits for ζ-globin, an endogenous, developmentally silenced, non-β-like globin.

A post-transcriptional process contributes to efficient γ-globin gene silencing in definitive erythroid cells

Objectives:  The expression of human γ 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 γ‐globin mRNA.

Functional effects of replacing human α‐ and β‐globins with their embryonic globin homologues in defined haemoglobin heterotetramers

Embryonic‐ and adult‐stage globin subunits assemble into haemoglobin (Hb) heterotetramers that are expressed at low levels throughout human intrauterine development. These haemoglobins differ from adult Hb A (α2β2) by the substitution of embryonic ζ for adult α globin (Hb ζ2β2), or embryonic ε for adult β globin (Hb α2ε2). Several key physiological properties of these ‘semiembryonic’ haemoglobins remain undefined, as ethical and methodological considerations have limited their availability from both human sources and conventional expression systems. The current study attempts to estimate how the physiological properties of semiembryonic and adult haemoglobins may differ, by determining whether the O2‐binding characteristics of hybrid human/mouse haemoglobins change when human α‐ or β‐globin subunits are replaced by human embryonic ζ‐ or ε‐globin subunits respectively. Each of the four human globins is expressed in transgenic mice that are nullizygous for either the endogenous mouse α‐ or β‐globin genes, resulting in the high‐level expression of haemoglobins that can be studied either in situ in intact erythrocytes or in vitro. We showed that the exchange of human ζ‐globin for human α‐globin chains increased haemoglobin O2 affinity, both in the presence and in the absence of 2,3‐bisphosphoglycerate (2,3‐BPG), and reduced the pH‐dependent shift in its oxygen equilibrium curve (Bohr effect). By comparison, hybrid haemoglobins containing either human ε‐globin or human β‐globin exhibited nearly identical O2‐binding properties, both in situ and in vitro, regardless of 2,3‐BPG levels or ambient pH. Neither the ζ‐for‐α nor the ε‐for‐β substitutions substantially altered binding affinity for 2,3‐BPG or cooperativity between globin subunits. These studies suggest that semiembryonic haemoglobins that assemble entirely from human subunits may exhibit properties that are similar to those of human Hb A.

Structure of fully liganded Hb ζ2β2s trapped in a tense conformation.

A variant Hb ζ2β2(s) that is formed from sickle hemoglobin (Hb S; α2β2(s)) by exchanging adult α-globin with embryonic ζ-globin subunits shows promise as a therapeutic agent for sickle-cell disease (SCD). Hb ζ2β2(s) inhibits the polymerization of deoxygenated Hb S in vitro and reverses characteristic features of SCD in vivo in mouse models of the disorder. When compared with either Hb S or with normal human adult Hb A (α2β2), Hb ζ2β2(s) exhibits 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 ζ2β2(s) that was expressed in complex transgenic knockout mice and purified from their erythrocytes is presented. When fully liganded with carbon monoxide, Hb ζ2β2(s) displays a central water cavity, a ζ1-β(s)2 (or ζ2-β(s)1) 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 ζ1β(s)1 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 ζ2β2(s) and, moreover, decouples tertiary- and quaternary-structural events that are critical to Hb ligand binding and allosteric function.