Destabilization of Human alpha -Globin mRNA by Translation Anti-termination Is Controlled during Erythroid Differentiation and Is Paralleled by Phased Shortening of the Poly(A) Tail*

(Received for publication, October 16, 1996, and in revised form, December 12, 1996)

Julia Morales Dagger , J. Eric Russell § and Stephen A. Liebhaber Dagger §

From the Dagger  Howard Hughes Medical Institute and § Departments of Genetics and Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

The extraordinary stability of globin mRNAs permits their accumulation to over 95% of total cellular mRNA during erythroid differentiation. The stability of human alpha -globin mRNA correlates with assembly of a sequence-specific ribonucleoprotein complex at its 3'-untranslated region. A naturally occurring anti-termination mutation, Constant Spring (CS), which permits ribosomes to enter the 3'-untranslated region of the alpha -globin mRNA, results in accelerated mRNA decay. To study the mechanism of this destabilization in vivo, we established transgenic mouse lines carrying the human alpha CS gene. Relative to wild-type human alpha -globin mRNA (alpha wt), alpha CS mRNA is destabilized in marrow erythroid cells. The poly(A) tails of both the alpha CS and alpha wt mRNAs show a periodicity of 20-25 nucleotides consistent with phased binding of poly(A) binding proteins. However, the mean size of poly(A) tails of the unstable alpha CS mRNA is significantly shorter than that of the alpha wt mRNA. Unexpectedly, the alpha wt and alpha CS mRNAs are of equal stability in peripheral reticulocytes, where their respective poly(A) tails shorten coordinately. These findings demonstrate a characteristic organization of the poly(A) tail on alpha -globin mRNA which is maintained during normal and accelerated decay, a correlation between poly(A) metabolism and anti-termination-mediated accelerated mRNA turnover, and a switch in the mechanism of mRNA decay during erythroid terminal differentiation.


INTRODUCTION

The phenotype and function of a cell are defined by the spectrum of its cytoplasmic mRNAs. The level of a specific mRNA is determined by the balance between its rates of synthesis and degradation. Recent studies in a variety of experimental systems highlight the central role of mRNA stability in the control of gene expression. The mechanisms that control this property appear complex and encompass distinct pathways for different subgroups of mRNAs (reviews in Refs. 1-3).

The half-lives of mRNAs in higher eucaryotes can differ by over 1000-fold, with a range of minutes to days (3). For example, the half-lives (t1/2) of c-myc and c-fos mRNAs are approximately 10-15 min (4) while the t1/2 of globin mRNA is more than 24 h (5). In some instances, mRNA stability may be a dynamic property that changes dramatically in response to developmental, environmental, or metabolic signals.

The stability of an mRNA is a function of its sequence and structure and may reflect interaction(s) of cis elements with trans-acting factors. For example, the stability of the transferrin receptor mRNA is controlled by intracellular concentration of iron; at low iron concentrations the transferrin receptor mRNA is quite stable, while a rise in cytosolic iron triggers its accelerated decay (6). This control is mediated by interaction of a specific series of secondary structures in the transferrin receptor mRNA 3'-untranslated region (3'-UTR)1 with an iron binding protein (6). In contrast to the transferrin receptor mRNA, the structural determinants that underlie the stability of most mRNAs are poorly defined.

mRNAs are degraded via a number of interrelated pathways, several of which have been defined in yeast (2). A common degradation pathway is initiated by shortening of the poly(A) tail, followed by removal of the 7-methylguanosine cap structure. The deadenylated and decapped mRNA is then degraded by a 5' right-arrow 3' exonuclease (7, 8). This pathway implies that there is communication between the 5'- and 3'-termini of the mRNA; the mechanism(s) involved in this interaction is not known (9). In higher eucaryotes, deadenylation also frequently accompanies degradation of mRNA, although the mechanism of degradation subsequent to deadenylation remains to be established (10). Two other less common degradation pathways are independent of prior deadenylation: direct 5'-decapping triggered by nonsense mutations (11) and site-specific endonuclease cleavage in the 3'-UTR (12, 13). The general importance of these pathways, how each is controlled, and how they might be interrelated are questions that are presently under study.

The globin genes provide a unique model system with which to study mechanisms responsible for mRNA stability. During terminal erythroid differentiation, globin mRNAs accumulate to over 95% of total cellular mRNA (14). This remarkable enrichment of a single group of mRNAs relies on their unusually long half-lives as well as selective degradation of non-globin mRNAs (5, 15-17). The importance of mRNA stability to globin gene expression is clearly demonstrated by the phenotype resulting from the loss of this property. For example, a <UNL>U</UNL>AA right-arrow <UNL>C</UNL>AA anti-termination mutation in the alpha -globin gene (Constant Spring, alpha CS), results in mRNA destabilization and consequent loss of over 95% expression from the affected locus (18).

Recent studies have demonstrated that alpha CS mRNA is destabilized by the physical entry of ribosomes into its 3'-UTR, which is permitted by the CS anti-termination mutation. This finding suggested that alpha -globin mRNA contains a stability determinant in this region (19). Subsequent studies confirmed this hypothesis and identified the critical cis-determinant as a pyrimidine-rich motif in the 3'-UTR that functions in an erythroid-specific manner (20). The stability motif serves as an assembly site for a sequence-specific mRNA-protein (mRNP) complex (alpha -complex, 21). The presence of this complex correlates with alpha -globin mRNA stability; mutations that block assembly of the alpha -complex in vitro also destabilize the mRNA in vivo (20, 21). Although structural studies have identified one component of the alpha -complex (22), its overall composition remains to be established.

Although the importance of the alpha -complex to alpha -globin mRNA stability is apparent, the mechanism(s) of its stabilizing function remains to be defined. One approach to this problem is to follow the pathway of globin mRNA degradation triggered by a mutation that interferes with normal stabilizing functions. In the present report we describe a fully physiologic in vivo model system to facilitate such an approach. Transgenic mice that express readily detectable levels of alpha CS mRNA were established. The stability and structure of alpha CS mRNA in these mice can be characterized and compared with wild-type human alpha -globin mRNA (halpha or alpha wt) expressed in a parallel set of transgenic lines. Using this model, we have focused our attention on the structure and dynamics of the poly(A) tail of halpha -globin mRNA in early erythroblasts in the bone marrow and in mature reticulocytes in the peripheral blood. Our findings demonstrate a phased organization of the poly(A) tail, which is independent of mRNA stability and maintained during differentiation. The data also reveal a substantial effect of the alpha CS mutation on its poly(A) tail size. Finally, we present evidence for a switch in the mechanism of halpha -globin mRNA decay during erythroid differentiation.


EXPERIMENTAL PROCEDURES

alpha CS Gene Construction

The alpha CS mutation was introduced by splice-overlap extension/polymerase chain reaction (Ref. 20 and included references) into the wild-type human alpha 2-globin gene carried as a 1.4-kb PstI insert in the pSP72Halpha 2 plasmid (23). Polymerase chain reaction amplifications were carried out using Vent polymerase (New England BioLabs) under the following conditions: 1 cycle (95 °C for 5 min, 57 °C for 15 s, and 73 °C for 25 s), 28 cycles (92 °C for 1 min, 57 °C for 15 s, and 73 °C for 25 s), 1 cycle (73 °C for 3 min). Primer name indicates its position relative to the transcriptional start site of the human alpha 2-globin gene. Primer SP72U hybridizes in the vector immediately 3' to the alpha -globin gene. Reaction R1 contained 10 ng of pSP72Halpha 2 template, 100 pmol each of primers 589 (5'CTGCACAGCTCCTAAGCCA3') and 717Reverse (5'GAGGCTTCCAGCTTGACGGTA3'), and yielded the expected 148-bp product. Reaction R2 contained 10 ng of template and 100 pmol each of primers 717 (5'TACCGTCAAGCTGGAGCCTC3') and SP72U (5'TAATACGACTCACTATAGGGA3') and yielded the expected 240-bp product. 1 µl each of reactions 1 and 2 were combined, heat-denatured, reannealed, and amplified with primers 589 and SP72U as above except that the extension time was increased to 30 s. The expected 368-bp splice-overlap extension product was digested with SphI and BstEII and ligated into the SphI/BstEII site of pSP72Halpha 2. Aliquots of the ligation mixture were electroporated into competent DH5alpha cells, and ampicillin-resistant colonies were screened by restriction analysis of plasmid DNA. Candidate clones were purified and sequenced to ensure sequence fidelity.

Generation of Transgenic Mice Expressing the alpha CS Gene

All transgenic mice were generated by the Transgenic Mouse Core Facility at the University of Pennsylvania School of Medicine. The alpha CS gene was released from its host pSP72 vector by EcoRI digest and introduced into the EcoRI site of the µLCR/pSP72 vector (23). The 8.0-kb µLCR/alpha CS fragment released by EcoRV/SalI digestion was purified from a 0.6% agarose gel over an Elutip column according to standard protocol (Schleicher & Schuell). The purified DNA was taken up in buffer (5 ng/µl) for injection into fertilized mouse oocytes (23). Transgenic alpha CS mice were identified by dot blot analysis of tail DNA using a beta -globin µLCR HSII probe. Six transgenic mouse lines carrying the normal alpha -globin transgene have been previously described (23); four additional lines constructed in the same manner were added for this study. Transgene copy numbers for alpha wt and alpha CS lines were in the same range. Transgene copy number was established by Southern analysis of EcoRI-digested tail DNA from F1 pups. A human alpha -globin promoter probe (0.6-kb NcoI fragment) detects a 23-kb fragment in the human genome and a 1.5-kb halpha -globin transgene fragment. The amount of DNA loaded on each lane was normalized using a probe to the 3'-flanking region of the endogenous mouse zeta -globin gene (2.3-kb BamHI/EcoRI fragment), which detects a 4.5-kb fragment of the mouse genome.

RNA Preparation

Adult transgenic mice were rendered anemic by three intraperitoneal injections of acetyl-2-phenylhydrazine (Sigma, 40 mg/g of body weight) over a 36-h period. Mice were decapitated 4.5 days after the initial injection, and blood was collected into heparinized (20 units/ml) phosphate-buffered saline (PBS). Femoral and tibial marrow was flushed into heparinized PBS using a 23-gauge needle. Total RNA was isolated from each tissue by lysis in 5 M guanidine thiocyanate solution (50 mM Tris, pH 7.4, 10 mM EDTA, 700 mM beta -mercaptoethanol, 1% sarcosyl) and centrifugation through a 5.7 M cesium chloride cushion at 42,000 rpm for 16 h in a TL55 rotor (Beckman). The RNA pellet was resuspended in 10 mM Tris, pH 7.4, 1 mM EDTA, 1% SDS, extracted twice with phenol/chloroform/isoamyl alcohol, and ethanol-precipitated. RNA purity and concentration were determined by A260/280 measurements.

In Vitro Culture of Peripheral Blood Reticulocytes

Heparinized blood from phenylhydrazine-treated mice was collected as described above, and cells were sedimented by centrifugation for 5 min at 1000 × g, washed in ice-cold PBS, resuspended in minimum essential medium alpha  supplemented with 10% fetal bovine serum, and incubated at 37 °C in 5% CO2. During each incubation, cell viability was verified by minimal release of hemoglobin into the media. Cell aliquots harvested at 0, 24, and 48 h were washed twice in PBS, and total RNA was isolated as described (24). To determine the rate of decay of mouse alpha -globin mRNA in cultured reticulocytes, cell number for each aliquot was determined, and RNA was isolated in the presence of a trace amount of [35S]UTP-labeled glyceraldehyde-3-phosphate dehydrogenase mRNA (specific activity, 700,000 cpm/µg) to control for RNA extraction efficiency. The amount of malpha -globin mRNA was determined per 5 × 105 cells by RNase protection assay.

RNase Protection Assay

Antisense RNA probes complementary to the first exon-encoded region of the human alpha -globin mRNA and the second exon-encoded region of the mouse alpha -globin mRNA were synthesized from plasmids pGEM3alpha H and pGEM3alpha M (23). Human alpha -globin (halpha -globin) probe was synthesized at 10 times the specific activity of the malpha -globin probe by adjusting the amount of [alpha -32P]CTP in the transcription mix (Maxiscript transcription kit, Ambion). Total RNA isolated from mouse bone marrow (500 ng of total RNA) or reticulocytes (100 ng of total RNA) was mixed with an excess of halpha - and malpha -globin RNA probes in 20 µl of hybridization buffer (80% formamide, 40 mM PIPES, pH 6.4, 400 mM NaCl, 1 mM EDTA), denatured at 75 °C for 15 min, and then hybridized at 52 °C for 16 h. Excess probe and unhybridized RNA were then digested by addition of 4 µg of RNase A and 200 ng of RNase T1 in a total of 200 µl of buffer (300 mM NaCl, 10 mM Tris, pH 7.4, 5 mM EDTA) and incubated for an additional 20 min at room temperature. The reaction was terminated by the addition of 17 µl of 1:4 mix of proteinase K (10 mg/ml) and SDS (10%), phenol-extracted, ethanol-precipitated, and resolved on an 8% acrylamide, 8 M urea gel. Signal intensity was quantified on a PhosphorImager with ImageQuant software (Molecular Dynamics).

Poly(A) Tail Analysis

Poly(A) tail length was determined by a site-specific RNase H cleavage assay (7). Total RNA (0.1-1 µg) was hybridized to oligonucleotide 662 (5'AACTTGTCCAGGGAGGCGTGC3') and digested with 0.05 units/µl RNase H in 20 µl of 20 mM Tris, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol, 30 mg/ml bovine serum albumin at 30 °C for 1 h. The reaction was terminated by phenol extraction and ethanol precipitation and then electrophoresed on a 6% acrylamide, 8 M urea gel. The RNA fragments were electrotransferred onto a nylon membrane (Nytran, Schleicher & Schuell). The membrane was hybridized 16 h at 42 °C in 50% formamide, 5 × SSC, 0.1% SDS, and 5 × Denhardt's reagent with 106-107 cpm of probe/ml. Blots were then washed in 0.1 × SSC, 0.1% SDS at 65 °C, exposed, and scanned on a PhosphorImager. The probe, which corresponds to a 109-bp fragment of the halpha -globin 3'-UTR, was amplified using primer HindIII/726 (5'TGGAAGCTTGCTGGAGCCTCGGTAGCCGT3') and primer EcoRI/833 (5'CCGAATTCGCCGCCCACTCAGACTTAT3') in a polymerase chain reaction containing 20 ng of pSP6Halpha 2FL template (alpha 2-globin cDNA; 25), 2.5 mM MgCl2, 0.2 mM each dATP, dTTP, and dGTP, 0.08 mM dCTP, 2 µCi of [alpha -32P]dCTP (Amersham Corp.), and 2.5 units of Taq polymerase (Perkin-Elmer), for 1 cycle (95 °C × 5 min, 56 °C × 15 s, and 72 °C × 35 s) and 30 cycles (95 °C × 1 min, 56 °C × 15 s, and 72 °C × 35 s). The synthesized probe was purified on a G50 spin column (Boehringer Mannheim) prior to use.

Western Blot Analysis and Gel Shift Assay

Cytoplasmic extracts were obtained by lysis of cells in 10 mM Hepes, pH 7.5, 10 mM KCl, 3 mM MgCl2, 1 mM dithiothreitol, 0.2% Triton X-100, 0.05% deoxycholate, 0.2 mg/ml heparin, and anti-proteases (0.2 µg/ml leupeptin, 0.2 µg/ml pepstatin, and 10 µg/ml aprotinin). Nuclei and cell debris were pelleted at 10,000 × g for 10 min. Supernatant protein concentration was estimated as described (26). Standard protocols were followed for Western analysis and enhanced chemiluminescence (Amersham Corp.) using polyclonal chicken anti-alpha CP antibody (1/10,000 dilution).2 Gel shift assay was performed as described (21). Briefly, an [alpha -32P]CTP-labeled halpha -globin 3'-UTR RNA probe (50,000 cpm/µl) was incubated with 50-450 µg of protein extract for 30 min at 20 °C; RNase T1 (1 units/µl) was added for an additional 10 min, and the complex was resolved on a 5% nondenaturing polyacrylamide gel.


RESULTS

Generation of Mice Expressing the Human alpha CS Transgene

In humans, the presence of the CS anti-termination mutation in the alpha -globin mRNA results in accelerated message decay. To establish an in vivo model system in which to study the mechanism(s) of this accelerated decay, transgenic mouse lines that expressed the human alpha CS gene were generated. An alpha CS gene was constructed by introducing the CS mutation into the alpha wt globin gene by splice-overlap extension (see "Experimental Procedures"). The alpha CS gene was then ligated to a DNA fragment containing all four hypersensitive sites of the human beta -globin locus control region (27) to ensure erythroid-specific high level transgene expression (23). Five alpha CS transgenic lines were established and the transgenes mapped in F1 progeny from each line to certify integrity and to establish copy number (Fig. 1B). For the study of alpha wt mRNA stability, mice from previously described transgenic lines were utilized. The alpha CS and alpha wt transgenes are identical except for the single base substitution at the translation termination codon (Fig. 1A), and the transgene copy numbers are in the same range (23).


Fig. 1. Generation of mouse lines carrying the human alpha CS transgene. A, the alpha CS transgene. The human alpha -globin gene comprises three exons (filled boxes) and two introns (open boxes). The transcriptional initiation site is marked by the angled arrow. The alpha CS mutation is a single base substitution (<UNL>U</UNL>AA right-arrow <UNL>C</UNL>AA) at the translation termination codon in the third exon. The alpha CS transgene is linked to the beta -globin µLCR to ensure high levels of erythroid cell-specific expression in transgenic mice. B, Southern blot analysis of five alpha CS transgenic lines. The transgene copy number, calculated from the relative signal intensities of the 1.5- and 4.5-kb bands (see "Experimental Procedures"), is shown below each respective lane. Normal mouse and human genomic DNA samples were run in parallel control lanes.
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The CS Mutation Destabilizes alpha -Globin mRNA in Transgenic Mice

The first goal was to demonstrate that the CS mutation destabilizes the halpha -globin mRNA in mouse erythroid tissue. The relative stabilities of alpha wt and alpha CS mRNAs were compared using an RNase protection assay that takes advantage of the natural transcriptional silencing that occurs in bone marrow during erythroid differentiation. The ratio of transgene halpha - to endogenous malpha -globin mRNA in the bone marrow and in the peripheral blood reticulocytes determines two points on an mRNA decay curve (normalized stability, ((halpha /malpha )peripheral blood/ (halpha /malpha )bone marrow)). A representative study is shown in Fig. 2A, and the results from the 10 alpha wt globin lines and the five alpha CS lines are shown in Fig. 2B. The average value of 0.85 for alpha wt mRNA indicates that its stability is equivalent (within experimental error) to that of the endogenous malpha -globin mRNA. The mean value of 0.2 for alpha CS mRNA indicates that it is highly unstable relative to both malpha - and halpha wt-globin mRNAs. The 3-fold spread in the normalized stabilities among the alpha wt lines does not adversely affect the high significance of the difference between alpha wt and alpha CS stability (see "Discussion"). Therefore, this transgenic model system accurately reproduces the destabilization of the mutant alpha CS mRNA observed in humans.


Fig. 2. The CS mutation destabilizes alpha -globin mRNA in transgenic mice. A, analysis of halpha -globin mRNA stability. Total mRNA was isolated from bone marrow (BM) and peripheral blood (PB) of phenylhydrazine-treated mice transgenic for the human alpha wt or alpha CS genes, and the levels of halpha - and malpha -globin mRNA were determined by quantitative RNase protection assay. The position and identity of each protected fragment is indicated (halpha and malpha ). The specific lines used in this representative study are shown above the respective lanes. B, normalized stabilities of the alpha wt and alpha CS mRNAs. Each symbol represents the mean value for an independent line as determined from analysis of two or more mice. Solid bars represent the average normalized stabilities of the transgenic mRNAs, and the dashed line indicates the stability of endogenous malpha -globin mRNA (defined as 1.0).
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The Poly(A) Tail of halpha -Globin mRNA Is Organized in Increments of 20-25 Nucleotides and Shortens during Erythroid Maturation

The alpha wt and alpha CS transgenic mRNAs were studied for the size and organization of their poly(A) tails. If accelerated decay were linked to deadenylation, we might be able to detect a difference in the poly(A) profile of the unstable alpha CS mRNA when compared with the stable alpha wt mRNA. The size of the human alpha -globin mRNA poly(A) tail was determined after site-specific RNase H cleavage of total RNA from bone marrow or peripheral blood (Fig. 3A). The digested fragments were separated by denaturing polyacrylamide gel electrophoresis and the 3'-terminal fragment was visualized by Northern blot analysis using a halpha -globin mRNA 3'-UTR probe. Typical profiles for alpha wt and alpha CS globin poly(A) tail are shown in Fig. 3B. In a parallel reaction, oligo(dT) was also added to the RNase H digestion, in order to generate a fully deadenylated 3'-terminal fragment (lane dT, Fig. 3B). The densitometric profiles of single lanes on the autoradiograph reflect the spectrum of poly(A) tail lengths in each sample (Fig. 3C).


Fig. 3. The poly(A) tails of halpha -globin mRNA are present in incremental sizes and are abnormally short in alpha CS mRNA. A, targeted cleavage of halpha -globin mRNA. The diagram indicates the position of the antisense oligonucleotide used to target RNase H digestion and the position of the probe used for Northern hybridization. B, poly(A) tail analysis of alpha wt and alpha CS mRNAs in marrow and peripheral blood erythroid cells. Fragment lengths were calculated using a DNA ladder (not shown). Poly(A) tail length of a particular fragment was determined by subtracting the length of the fully deadenylated fragment (dT lane) from its total length. C, scanning densitometry of gel (B). The positions of each of the sized peaks is indicated, as is the position of the deadenylated fragment. The direction of electrophoretic migration is noted below the scan.
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Analysis of alpha wt mRNA revealed that poly(A) tail lengths have a periodicity representing increments of 20-25 nucleotides (Fig. 3B). In the bone marrow, the alpha wt mRNA contains a dominant A60 peak flanked by peaks of lower intensity that correspond to A85 and A40 (Fig. 3B). By comparison, the poly(A) tail of the halpha -globin mRNA in peripheral blood reticulocytes is partitioned into four peaks with an overall shift to a smaller mean size; although A60 is still the dominant peak, A85 decreases in intensity, and a significant A20 peak appears. Of note, we do not detect any evidence of fully deadenylated mRNA. These analyses of the alpha wt globin mRNA indicate that the poly(A) tail is organized in a modular fashion and that the mean poly(A) tail size decreases as bone marrow erythroid cells mature into peripheral reticulocytes. Therefore, alpha -globin mRNA poly(A) tail phasing is maintained despite its age-related shortening in differentiating erythroid cells.

The CS Mutation Is Linked to an Accelerated and Phased Shortening of Poly(A) Tail

The size distribution of the alpha CS mRNA poly(A) tail was determined using the RNase H assay. The fully deadenylated alpha CS fragment migrated at the same position as the alpha wt fragment (data not shown), indicating that the 3' end of both mRNAs were processed identically. In bone marrow, the alpha CS mRNA poly(A) tail was significantly shorter than the alpha wt and was present in two major peaks of A60 and A40 (Fig. 3, B and C). In reticulocytes, the alpha CS A40 peak became more prominent. In contrast to the alpha wt globin mRNA, there was no significant A20 signal. Thus, like the alpha wt mRNA, the alpha CS mRNA shows age-related, phased shortening of its poly(A) tail. However, the distribution of the poly(A) tail size on the alpha CS mRNA is significantly different than on the alpha wt mRNA in both the bone marrow and in peripheral reticulocytes.

alpha wt and alpha CS mRNAs Are of Equal Stability in Late Stage Reticulocytes

Under normal conditions, all erythroid mRNAs undergo final degradation over a 2-3-day period in peripheral blood reticulocytes. This terminal event in erythroid cell maturation may involve stability mechanisms and determinants distinct from those that favor rapid accumulation of globin mRNAs during the earlier phases of erythroid maturation. To directly assess this possibility, we followed mRNA decay during a 48-h ex vivo incubation of peripheral blood reticulocytes. Reticulocyte viability appeared fully maintained during incubation. Endogenous malpha -globin mRNA levels fell by 70% during the 48-h incubation (Fig. 4A), indicating that this ex vivo incubation reproduces the reticulocyte mRNA clearance observed in vivo (28).


Fig. 4. alpha wt and alpha CS mRNA decay at equivalent rates in ex vivo cultured reticulocytes. Peripheral blood was collected from phenylhydrazine-treated mice and cultured ex vivo for 48 h. Total RNA was isolated from aliquots harvested at 0, 24, and 48 h. A, murine alpha -globin mRNA content per cell aliquot as quantified by RNase protection assay. Values for 24 and 48 h incubations were expressed as a fraction of the t0 value. The mean and standard error is derived from the study of reticulocytes from four nontransgenic mice. B, RNase protection assay of ex vivo incubated reticulocytes from transgenic mice. RNA isolated from alpha wt and alpha CS reticulocytes was analyzed as described in Fig. 2. The positions of malpha - and halpha -protected fragments are noted. The band migrating between malpha and halpha bands in alpha CS lanes, also present in the reticulocyte control (right lane), is due to protection of an unidentified endogenous mouse RNA. C, signals from the gel (B) were quantitated on PhosphorImager. Levels of transgenic halpha -globin mRNA (alpha wt or alpha CS) were estimated relative to endogenous malpha -globin mRNA; values for 24 and 48 h incubation were expressed as a fraction of the t0 value. The data shown are averaged from studies of four alpha wt transgenic lines and three alpha CS lines.
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The levels of transgenic alpha wt and alpha CS mRNAs were measured relative to endogenous mouse alpha -globin mRNA in ex vivo incubated reticulocytes over a 48-h period. A typical RNase protection assay is shown in Fig. 4B, and average stabilities from studies of four alpha wt and three alpha CS lines are plotted in Fig. 4C. Consistent with the accelerated decay of the alpha CS mRNA in bone marrow erythroid cells, the initial levels of alpha CS mRNA were considerably lower than alpha wt mRNA levels. Surprisingly, however, the stabilities of the alpha CS and the alpha wt mRNAs were equivalent. Thus the alpha CS mRNA is unstable relative to the alpha wt mRNA in the bone marrow, but the alpha wt and alpha CS mRNAs decay at comparable rates in peripheral reticulocytes.

Poly(A) Tails of alpha wt and alpha CS Globin mRNAs Shorten Coordinately in Peripheral Reticulocytes

The kinetics of poly(A) tail shortening were determined in the ex vivo incubated reticulocytes (Fig. 5). The alpha wt globin mRNA shortened over the 48-h incubation from an initial multipeak distribution to a single intense peak of A20. At the beginning of the incubation period, alpha CS mRNA was distributed into two peaks, A60 and A40. Like the alpha wt mRNA poly(A) tails, the alpha CS poly(A) tails that remain after an additional 48 h were shortened to a single A20 peak. Thus for both mRNAs there is a progressive shortening of the poly(A) tail to a minimal size of A20. Whether deadenylation precedes or parallels mRNA degradation during late reticulocyte differentiation is unknown. The appearance of the A20 peak in the alpha CS mRNA poly(A) tail, in coordination with a equivalent rate of decay for both alpha wt and alpha CS mRNA, suggests a switch in the degradation pathway of globin mRNA in the late stages of reticulocyte differentiation.


Fig. 5. Poly(A) tails of alpha wt and alpha CS-globin mRNAs shorten coordinately in ex vivo cultured reticulocytes. A, poly(A) tail analysis of mRNA from alpha wt and alpha CS transgenic mice reticulocytes incubated ex vivo for 48 h. Analysis was performed as described in Fig. 3. B, scanning densitometry of single lanes from gel (A). The position of each peak is indicated, as is the position of the deadenylated 3'-terminal fragment. The direction of electrophoretic migration is noted below the scan.
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The Level of an Essential Subunit of the alpha -Globin Stabilizing Complex Decreases During Erythroid Terminal Differentiation

alpha CS is degraded faster than alpha wt mRNA in marrow erythroid cells. The same accelerated decay of alpha CS mRNA has been noted in cultured MEL cells, which represent an early stage in terminal erythroid differentiation (19). By comparison, both mRNAs are equally stable in peripheral reticulocytes. The stability of alpha wt mRNA in early erythroblasts (MEL cells) is dependent upon formation of an RNP complex on its 3'-UTR (21). The convergence of alpha wt and alpha CS mRNA stabilities during terminal erythroid differentiation might therefore reflect a decrease in the ability of mature cells to assemble the alpha -complex and selectively protect the alpha wt mRNA. To test this hypothesis, we compared the levels of alpha CP, a 39-kDa RNA-binding protein that is an essential subunit of the alpha -complex (22), in MEL cells and in peripheral reticulocytes from phenylhydrazine-treated mice (Fig. 6, A and B). The limiting amount of mouse bone marrow and the necessity to separate erythroid from nonerythroid cells prior to assay (alpha CP is expressed ubiquituously (21)) precluded use of bone marrow cells as a source of early erythroid cells for this purpose. The signal observed by Western analysis in 450 µg of reticulocyte proteins is equivalent to that of 15 µg of MEL cell proteins. A slightly slower migration for alpha CP is observed in reticulocyte extract. Whether this is a result of a posttranslational modification or an aberrant migration pattern due to sample overloading is unknown. The alpha CP in the reticulocyte extracts retains the ability to bind to polyribocytidine (data not shown). The capacity of protein extracts from reticulocytes and MEL cells to assemble the alpha -complex was tested in parallel (Fig. 6C). The reticulocyte extract can assemble a normal alpha -complex, as judged by its comigration with the alpha -complex from MEL extract and its poly(C) sensitivity. However, the efficiency of complex formation is markedly reduced in comparison with the MEL cell extract. Thus, levels of alpha CP and alpha -complex decrease sharply in erythroid cells undergoing terminal differentiation.


Fig. 6. alpha CP is less abundant in murine reticulocytes than in MEL cells. A, silver stain of a 12% SDS-polyacrylamide gel. Lanes contain increasing amounts of cytoplasmic extract from MEL cells (left) or from mouse peripheral blood reticulocytes (right). Molecular weight markers are indicated to the left of the gel. The position of globin proteins is indicated. B, Western blot analysis of gel (A) using an anti-alpha CP antibody. C, gel shift assay using a [32P]alpha -globin 3'-UTR probe and the indicated amount of protein extract. The position of the alpha -complex is indicated by a bracket.
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DISCUSSION

The alpha -globin gene encodes an mRNA whose extraordinary stability has been demonstrated in multiple experimental systems (5, 14, 15, 17, 20). This stability is dependent upon the assembly of an mRNP complex on its 3'-UTR (21). Although the cis and trans elements of the alpha -complex are under study, the mechanisms by which the mRNA is stabilized in the developing erythroblast and subsequently cleared from the terminally differentiating peripheral reticulocyte are poorly understood. The generation of transgenic mice that express the halpha -globin (alpha wt) mRNA or the mutant alpha CS permits detailed study of these pathways in vivo.

The alpha CS Transgenic Mouse Model

Of the one hundred or more mutations of the alpha -globin gene documented in alpha -thalassemic individuals, the CS defect is unique in its direct destabilization of the mRNA (29, 30). The accelerated decay of alpha CS mRNA in mice appears to recapitulate its observed instability in humans. The two-point decay curve that was determined in vivo in transgenic mice demonstrated a 4-5-fold lower stability for alpha CS mRNA than for alpha wt-globin mRNA (Fig. 2). Cells from the bone marrow compartment, which include transcriptionally active erythroblasts as well as transcriptionally inactive normoblasts and reticulocytes, were compared in this analysis with a population of purely posttranscriptional, nonnucleated reticulocytes from the peripheral circulation. The marked fall in the alpha CS mRNA levels between these two compartments parallels observations in affected humans. In the bone marrow of individuals heterozygous for the alpha CS mutation, alpha CS and alpha wt mRNA levels are equivalent, whereas in peripheral reticulocytes alpha CS mRNA is nearly undetectable despite the continued presence of alpha wt mRNA (29).

The measurement of mRNA stability in the intact mouse as described in this report is noteworthy because the environment is entirely physiologic. Our approach avoids the use of transcriptional inhibitors such as actinomycin D, which can have a severe impact on cellular physiology and has been shown, in some cases, to paradoxically stabilize mRNA (31). The intra-assay variability in our study (multiple assays on an individual sample) is small, as is the variability among mice from the same line (12%, data not shown). However, we observed a 3-fold spread in the stability of alpha wt mRNA among individual transgenic lines (Fig. 2B). This variability does not appear to relate to transgene copy number, age, or sex of the mice. Therefore, although the assay itself is highly reliable, an additional biologic variable that we do not yet understand contributes to the stability of halpha -globin mRNA in the transgenic setting. It will be of great interest to identify this variable(s) in future studies. The alpha CS transgenic lines do not show such a variability in their normalized stability. Despite the variability between different alpha wt transgenic lines, the overall difference between the stability of alpha wt and alpha CS mRNA remains highly significant.

Poly(A) Tails of alpha -Globin mRNA Display a Discontinuous Size Distribution That May Reflect a Phased Array of Poly(A) Binding Proteins

Analysis of the alpha wt mRNA revealed that the size distribution of its poly(A) tails is not continuous but rather peaks with a periodicity of 20-25 nucleotides. This spacing suggests a role for the poly(A) binding protein (PABP) in the protection and/or organization of the poly(A) tail. Mammalian PABP is a 70-kDa protein that is involved in mRNA stability and translation (9, 32-34). PABP monomers bind poly(A) with a footprint of 20-25 residues (35, 36). The observed poly(A) tail profile for the alpha -globin mRNA (Fig. 3) indicates than the PABPs are "anchored" at fixed positions relative to the mRNA, i.e. they are phased with regard to the terminus of the 3'-UTR.

The demonstration of multiple peaks of poly(A) tail size is scarce in the literature. Previous studies on globin mRNA of other species (37, 38) have shown a similar pattern. One other report shows discrete poly(A) tail lengths for long-lived ribulose-bisphosphate carboxylase/oxygenase small subunit 2 mRNA in Chlamydomonas (34). Poly(A) tail phasing may be important for maintaining stability of long-lived mRNAs. Using an in vivo system, we have demonstrated that the poly(A) phasing pattern is dynamic. Specifically, we show that the 20-25 nucleotide spacing in poly(A) tail size was maintained during erythroid differentiation despite the overall poly(A) tail shortening of the human alpha -globin. This observation suggests that PABP associates with the poly(A) tail throughout terminal erythroid differentiation, and its shortening by integral 20-25 nucleotide units may reflect rapid degradation of deprotected segments as PABP monomers are released from the 3'-terminus.

Despite the accelerated shortening of its poly(A) tail, phasing at intervals of 20-25 nucleotides is maintained for the alpha CS mRNA. Because accelerated decay of the alpha CS mRNA is likely to reflect the displacement of the alpha -complex by the translating ribosome (19), PABP phasing would not appear to be dependent on the presence of the alpha -complex. However, the alpha -complex might serve to stabilize the poly(A) tail, perhaps by direct physical interaction with PABP or by strengthening the interaction between PABP and the poly(A) tail.

Selective Degradation of alpha CS mRNA Occurs Early but Not Late in Erythrocyte Differentiation

An unexpected observation in the present study was that the alpha CS and alpha wt mRNAs were degraded at equivalent rates in peripheral reticulocytes. This contrasts with the accelerated decay of alpha CS mRNA compared with alpha wt mRNA in the less differentiated erythroid cells both in vivo (this report) and in cultured mouse erythroblast cells (MEL, Ref. 20). The 60 h t1/2 of the alpha -globin mRNA in MEL cells (39) is shortened to 20 h by the CS mutation.3 Since the rate of decay of both alpha CS and alpha wt mRNAs paralleled that of the endogenous mouse alpha -globin mRNA (Fig. 4), we estimated their t1/2 in reticulocytes to be approximately 24 h. This change in the relative stability of the alpha CS and alpha wt mRNA may reflect a difference between the mechanism that determines alpha wt mRNA stability in early and late erythroid differentiation. In early erythroid cells, globin mRNAs are preferentially stabilized to facilitate their rapid accumulation and permit synthesis of high levels of globin protein. In contrast, in reticulocytes all cellular organelles, membrane remnants, and RNAs, including alpha -globin mRNAs, are rapidly cleared to generate a maximally deformable and functional erythrocyte. The estimated half-lives of the globin mRNAs are consistent with the time frame required for this clearance. We therefore speculate that the mechanism necessary to selectively accumulate globin mRNA is operative only in early stages of differentiation and that a more general mechanism is responsible for clearance of RNA from peripheral reticulocytes. To support this hypothesis, we determined that the concentration of alpha CP, one component of the RNP complex necessary for stabilization of alpha wt in early erythroblasts, is markedly diminished in circulating reticulocytes. In parallel, the ability of cell extracts to assemble the alpha -complex decreases from early erythroblasts to reticulocytes (Fig. 6).

A Model for the Regulation of alpha -Globin Stability in Differentiating Erythroid Cells

The stability of the alpha -globin mRNA is determined by the assembly of an mRNP complex on the 3'-UTR (20, 21). Based upon our data, we propose a model for the regulation of alpha -globin mRNA stability in differentiating erythroid cells (Fig. 7). mRNA bound to the alpha -complex is stabilized in early erythroid cells. beta -Globin mRNA and other mRNAs that are present at the reticulocyte stage may be stabilized by a related or different mechanism. Other mRNAs, whose decay is known to be accelerated during terminal erythroid differentiation (16, 17), are hypothesized to lack a stabilizing complex. As modeled by the alpha CS mRNA, accelerated decay is accompanied by accelerated shortening of the poly(A) tail. In the terminal stages of reticulocyte maturation, selectivity of mRNA decay is lost, and all mRNAs, including alpha wt mRNA, are rapidly cleared from the cell. This may reflect either a switch in the degradation pathway or a change in the manner in which specific mRNAs are protected. For instance, terminal decay might be accelerated by the loss of one or more components of the alpha -complex as detailed above and demonstrated in Fig. 6. Our model suggests that the alpha -complex no longer protects alpha -globin mRNA in terminally differentiated reticulocytes. Whether this is a solely passive effect of the decrease in the components of the alpha -complex or reflects a more active control of alpha -complex assembly remains to be determined.


Fig. 7. Regulation of human alpha -globin stability during erythroid cell maturation: a model. In early erythroblasts the globin genes are actively transcribed. At this stage the alpha -complex forms on the 3'-UTR of the halpha -globin mRNA, stabilizing the mRNA, and facilitating its accumulation to high concentrations. This stabilization may occur via an interaction between the alpha -complex and the poly(A)-PABP complex, protecting the poly(A) tail from degradation. Mutations (for example CS) that prevent formation or function of the alpha -complex reduce mRNA stability and expose the poly(A) tail to accelerated shortening. As the erythroblast differentiates and accumulates a critical level of globin mRNA, all transcription is silenced. At this point the levels of alpha CP fall and the alpha -complex dissociates from the 3'-UTR. Both the normal (wt) and mutant (CS) alpha -globin mRNAs now lack the alpha -complex and have equivalent stability. The poly(A) tails of both are now shortened at an equal rate with consequent clearance of the mRNA.
[View Larger Version of this Image (29K GIF file)]



FOOTNOTES

*   This work was supported in part by National Institutes of Health Grants HL38632 (to S. A. L.) and K11-HL02623 (to J. E. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
   Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Rm. 428 CRB, 415 Curie Blvd., Philadelphia, PA 19104.
1   The abbreviations used are: UTR, untranslated region; CS, Constant Spring; bp, base pair(s); kb, kilobase pair(s); PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid; wt, wild type; h, human; m, mouse; RNP, ribonucleoprotein; PABP, poly(A) binding protein.
2   M. Kiledjian and S. A. Liebhaber, unpublished results.
3   J. Morales, J. E. Russell, and S. A. Liebhaber, unpublished results.

Acknowledgments

We thank Alice Lee for technical assistance and members of the laboratory for helpful discussions. We also thank James Alwine and Michael Malim for critical reading of the manuscript.


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