(Received for publication, October 16, 1996, and in revised form, December 12, 1996)
From the Howard Hughes Medical Institute and
§ Departments of Genetics and Medicine, University of
Pennsylvania School of Medicine,
Philadelphia, Pennsylvania 19104
The extraordinary stability of globin mRNAs
permits their accumulation to over 95% of total cellular mRNA
during erythroid differentiation. The stability of human -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
-globin
mRNA, results in accelerated mRNA decay. To study the mechanism
of this destabilization in vivo, we established transgenic
mouse lines carrying the human
CS gene. Relative to
wild-type human
-globin mRNA (
wt),
CS mRNA is destabilized in marrow erythroid cells.
The poly(A) tails of both the
CS and
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
CS mRNA is
significantly shorter than that of the
wt mRNA.
Unexpectedly, the
wt and
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
-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.
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
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 AA
AA anti-termination
mutation in the
-globin gene (Constant Spring,
CS),
results in mRNA destabilization and consequent loss of over 95%
expression from the affected locus (18).
Recent studies have demonstrated that 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
-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 (
-complex, 21).
The presence of this complex correlates with
-globin mRNA
stability; mutations that block assembly of the
-complex in
vitro also destabilize the mRNA in vivo (20, 21).
Although structural studies have identified one component of the
-complex (22), its overall composition remains to be
established.
Although the importance of the -complex to
-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
CS mRNA were established. The
stability and structure of
CS mRNA in these mice can
be characterized and compared with wild-type human
-globin mRNA
(h
or
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 h
-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
CS mutation on its poly(A) tail size. Finally, we
present evidence for a switch in the mechanism of h
-globin mRNA
decay during erythroid differentiation.
The CS
mutation was introduced by splice-overlap extension/polymerase chain
reaction (Ref. 20 and included references) into the wild-type human
2-globin gene carried as a 1.4-kb PstI insert in the
pSP72H
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
2-globin gene. Primer SP72U
hybridizes in the vector immediately 3
to the
-globin gene.
Reaction R1 contained 10 ng of pSP72H
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 pSP72H
2. Aliquots of
the ligation mixture were electroporated into competent DH5
cells,
and ampicillin-resistant colonies were screened by restriction analysis
of plasmid DNA. Candidate clones were purified and sequenced to ensure
sequence fidelity.
All transgenic mice were generated by the Transgenic Mouse
Core Facility at the University of Pennsylvania School of Medicine. The
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/
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
CS mice were identified by dot blot analysis
of tail DNA using a
-globin µLCR HSII probe. Six transgenic mouse
lines carrying the normal
-globin transgene have been previously
described (23); four additional lines constructed in the same manner
were added for this study. Transgene copy numbers for
wt
and
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
-globin
promoter probe (0.6-kb NcoI fragment) detects a 23-kb
fragment in the human genome and a 1.5-kb h
-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
-globin gene
(2.3-kb BamHI/EcoRI fragment), which detects a
4.5-kb fragment of the mouse genome.
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 -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.
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 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
-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 m
-globin mRNA was
determined per 5 × 105 cells by RNase protection
assay.
Antisense RNA probes complementary
to the first exon-encoded region of the human -globin mRNA and
the second exon-encoded region of the mouse
-globin mRNA were
synthesized from plasmids pGEM3
H and pGEM3
M (23). Human
-globin (h
-globin) probe was synthesized at 10 times the specific
activity of the m
-globin probe by adjusting the amount of
[
-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 h
- and m
-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 length was determined by
a site-specific RNase H cleavage assay (7). Total RNA (0.1-1 µg) was
hybridized to oligonucleotide 662 (5AACTTGTCCAGGGAGGCGTGC3
) 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 h
-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 pSP6H
2FL template
(
2-globin cDNA; 25), 2.5 mM MgCl2, 0.2 mM each dATP, dTTP, and dGTP, 0.08 mM dCTP, 2 µCi of [
-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.
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-CP antibody (1/10,000 dilution).2 Gel shift
assay was performed as described (21). Briefly, an [
-32P]CTP-labeled h
-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.
In humans, the presence of the CS anti-termination
mutation in the -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
CS gene were generated.
An
CS gene was constructed by introducing the CS
mutation into the
wt globin gene by splice-overlap
extension (see "Experimental Procedures"). The
CS
gene was then ligated to a DNA fragment containing all four
hypersensitive sites of the human
-globin locus control region (27)
to ensure erythroid-specific high level transgene expression (23). Five
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
wt mRNA stability, mice from previously
described transgenic lines were utilized. The
CS and
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).
The CS Mutation Destabilizes
The first goal was to demonstrate that the CS mutation
destabilizes the h-globin mRNA in mouse erythroid tissue.
The relative stabilities of
wt and
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 h
-
to endogenous m
-globin mRNA in the bone marrow and in the
peripheral blood reticulocytes determines two points on an mRNA
decay curve (normalized stability, ((h
/m
)peripheral
blood/ (h
/m
)bone marrow)). A representative
study is shown in Fig. 2A, and the results
from the 10
wt globin lines and the five
CS lines are shown in Fig. 2B. The average
value of 0.85 for
wt mRNA indicates that its
stability is equivalent (within experimental error) to that of the
endogenous m
-globin mRNA. The mean value of 0.2 for
CS mRNA indicates that it is highly unstable
relative to both m
- and h
wt-globin mRNAs. The
3-fold spread in the normalized stabilities among the
wt
lines does not adversely affect the high significance of the difference
between
wt and
CS stability (see
"Discussion"). Therefore, this transgenic model system accurately
reproduces the destabilization of the mutant
CS mRNA
observed in humans.
The Poly(A) Tail of h
The wt and
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
CS mRNA when compared with the stable
wt mRNA. The size of the human
-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 h
-globin mRNA 3
-UTR probe. Typical profiles for
wt and
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).
Analysis of wt mRNA revealed that poly(A) tail
lengths have a periodicity representing increments of 20-25
nucleotides (Fig. 3B). In the bone marrow, the
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 h
-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
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,
-globin mRNA poly(A) tail phasing is maintained despite its
age-related shortening in differentiating erythroid cells.
The size distribution of the CS
mRNA poly(A) tail was determined using the RNase H assay. The fully
deadenylated
CS fragment migrated at the same position
as the
wt fragment (data not shown), indicating that the
3
end of both mRNAs were processed identically. In bone marrow,
the
CS mRNA poly(A) tail was significantly shorter
than the
wt and was present in two major peaks of
A60 and A40 (Fig. 3, B and
C). In reticulocytes, the
CS A40
peak became more prominent. In contrast to the
wt globin
mRNA, there was no significant A20 signal. Thus, like the
wt mRNA, the
CS mRNA shows
age-related, phased shortening of its poly(A) tail. However, the
distribution of the poly(A) tail size on the
CS mRNA
is significantly different than on the
wt mRNA in
both the bone marrow and in peripheral 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 m-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).
The levels of transgenic wt and
CS
mRNAs were measured relative to endogenous mouse
-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
wt and three
CS lines are plotted in Fig.
4C. Consistent with the accelerated decay of the
CS mRNA in bone marrow erythroid cells, the initial
levels of
CS mRNA were considerably lower than
wt mRNA levels. Surprisingly, however, the
stabilities of the
CS and the
wt
mRNAs were equivalent. Thus the
CS mRNA is
unstable relative to the
wt mRNA in the bone marrow,
but the
wt and
CS mRNAs decay at
comparable rates in peripheral reticulocytes.
The
kinetics of poly(A) tail shortening were determined in the ex
vivo incubated reticulocytes (Fig. 5). The
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,
CS mRNA was distributed into two peaks,
A60 and A40. Like the
wt
mRNA poly(A) tails, the
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
CS mRNA poly(A) tail, in
coordination with a equivalent rate of decay for both
wt
and
CS mRNA, suggests a switch in the degradation
pathway of globin mRNA in the late stages of reticulocyte
differentiation.
The Level of an Essential Subunit of the
CS is degraded faster than
wt mRNA in marrow erythroid cells. The same
accelerated decay of
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
wt mRNA in early erythroblasts (MEL cells) is
dependent upon formation of an RNP complex on its 3
-UTR (21). The
convergence of
wt and
CS mRNA
stabilities during terminal erythroid differentiation might therefore
reflect a decrease in the ability of mature cells to assemble the
-complex and selectively protect the
wt mRNA. To
test this hypothesis, we compared the levels of
CP, a 39-kDa
RNA-binding protein that is an essential subunit of the
-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
(
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
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
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
-complex was tested
in parallel (Fig. 6C). The reticulocyte extract can assemble
a normal
-complex, as judged by its comigration with the
-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
CP and
-complex decrease sharply in erythroid cells undergoing terminal differentiation.
The -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
-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 h
-globin
(
wt) mRNA or the mutant
CS permits
detailed study of these pathways in vivo.
Of the one
hundred or more mutations of the -globin gene documented in
-thalassemic individuals, the CS defect is unique in its direct
destabilization of the mRNA (29, 30). The accelerated decay of
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
CS mRNA than for
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
CS mRNA levels
between these two compartments parallels observations in affected
humans. In the bone marrow of individuals heterozygous for the
CS mutation,
CS and
wt
mRNA levels are equivalent, whereas in peripheral reticulocytes
CS mRNA is nearly undetectable despite the continued
presence of
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
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 h
-globin mRNA
in the transgenic setting. It will be of great interest to identify
this variable(s) in future studies. The
CS transgenic
lines do not show such a variability in their normalized stability.
Despite the variability between different
wt transgenic
lines, the overall difference between the stability of
wt and
CS mRNA remains highly
significant.
Analysis of the 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
-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 -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 CS
mRNA. Because accelerated decay of the
CS mRNA
is likely to reflect the displacement of the
-complex by the
translating ribosome (19), PABP phasing would not appear to be
dependent on the presence of the
-complex. However, the
-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.
An unexpected
observation in the present study was that the CS and
wt mRNAs were degraded at equivalent rates in
peripheral reticulocytes. This contrasts with the accelerated decay of
CS mRNA compared with
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
-globin mRNA in MEL
cells (39) is shortened to 20 h by the CS
mutation.3 Since the rate of decay of both
CS and
wt mRNAs paralleled that of
the endogenous mouse
-globin mRNA (Fig. 4), we estimated their
t1/2 in reticulocytes to be approximately 24 h.
This change in the relative stability of the
CS and
wt mRNA may reflect a difference between the
mechanism that determines
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
-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
CP, one component of the RNP complex necessary for
stabilization of
wt in early erythroblasts, is markedly
diminished in circulating reticulocytes. In parallel, the ability of
cell extracts to assemble the
-complex decreases from early
erythroblasts to reticulocytes (Fig. 6).
The stability of the -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
-globin mRNA stability in differentiating erythroid cells (Fig.
7). mRNA bound to the
-complex is stabilized in
early erythroid cells.
-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
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
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
-complex as detailed above and
demonstrated in Fig. 6. Our model suggests that the
-complex no
longer protects
-globin mRNA in terminally differentiated
reticulocytes. Whether this is a solely passive effect of the decrease
in the components of the
-complex or reflects a more active control
of
-complex assembly remains to be determined.
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.