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Blood, Vol. 93 No. 6 (March 15), 1999:
pp. 2111-2120
Identification of the Poly(C) Binding Protein in the Complex
Associated With the 3' Untranslated Region of Erythropoietin
Messenger RNA
By
Maria F. Czyzyk-Krzeska and
Amy C. Bendixen
From the Department of Molecular and Cellular Physiology, University
of Cincinnati, College of Medicine, Cincinnati, OH.
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ABSTRACT |
Hypoxia regulates expression of erythropoietin (EPO), a glycoprotein
that stimulates erythrocytosis, at the level of transcription and also
possibly at the level of messenger RNA (mRNA) stability. A
pyrimidine-rich region within the EPO mRNA 3' untranslated region was implicated in regulation of EPO mRNA stability element and shown to
bind protein factors. In the present study we wished to identify the
protein factor binding to the pyrimidine-rich sequence in the EPO mRNA
stability element. Using mobility shift assays, ultraviolet light
cross-linking, and sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE), and electroelution of protein factors from
the gel slices corresponding to the ribonucleoprotein complexes, we
found that two isoforms of a 40 kD poly(C) binding protein (PCBP, also
known as  CP or hnRNPE), PCBP1, and PCBP2 are
present in that complex. In Hep3B or HepG2 cells hypoxia induces neither expression of PCBP nor formation of the ribonucleoprotein complex associated with EPO mRNA that involves PCBP.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
ERYTHROPOIETIN (EPO) IS A GLYCOPROTEIN
synthesized and released from the kidney, which regulates
differentiation and maturation of progenitor cells toward erythrocytes,
leading to polycythemia during long-term adaptation to
hypoxia.1 EPO expression under normoxic conditions is
minimal, but increases during exposure to hypoxia in the
fibroblast-like type-I interstitial cells of the kidney cortex and
outer medulla,2 and also in the two cell lines derived from
liver tumors (Hep3B, HepG2) that express EPO in an
O2-regulated manner.3 EPO synthesis during
hypoxia is induced at the level of gene expression; in addition to
transcriptional induction,4 it may involve regulation of
EPO mRNA stability.5 Transcriptional regulation of EPO
induction by hypoxia has been studied extensively.6 In
contrast, much less is known about regulation of EPO messenger RNA
(mRNA) stability, primarily because EPO mRNA stability is affected both
by ongoing transcription and by protein synthesis.5
Analysis of the 3' untranslated region (3' UTR) of EPO mRNA
shows putative stability and instability elements. Deletion of the 186 bases of the conserved sequence from the distal 3' UTR of EPO mRNA increased the t1/2 from 2 hours to 15 hours.7 This indicates that the deleted fragment of
3' UTR may contain an RNA instability element, whereas the
remaining region, located 5' from the deleted region, may contain
an RNA stability element.7 Deletion of the 104 bases
immediately downstream from the EPO translation stop codon results in
destabilization of EPO mRNA from 7 hours to 2.6 hours, an indication
that this fragment may contain an RNA stability
element.8 These experiments, however, were performed in the
presence of actinomycin D, which nonspecifically stabilizes EPO
mRNA.5,8 Thus it is difficult to estimate the role of this
fragment of EPO 3' UTR in regulating EPO mRNA stability. This
putative EPO mRNA stability region previously was found to
bind cytoplasmic proteins.9,10 Ultraviolet (UV) light
cross-linking and sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) analysis of the EPO RNA-protein complexes
showed two bands of 70 and 135 to 140 kD.9 Except in the
brain and the spleen, however, the binding activity in most cell lines
and tissues studied was not induced by hypoxia.9 The
binding of these proteins to EPO mRNA was redox-sensitive and required
reduced thiol groups.10
Regulation at the level of mRNA stability during hypoxia occurs in the
case of tyrosine hydroxylase (TH), the rate-limiting enzyme in
catecholamine synthesis in pheochromocytoma-derived PC12 cells. Hypoxia
augments TH mRNA half-life twofold.11,15 This increase in
TH mRNA stability during hypoxia involves enhanced formation of a
ribonucleoprotein complex associated with an RNA stability element in
the 3' UTR of TH mRNA (hypoxia-inducible protein-binding
sequence, HIPBS).12-15 The binding proteins are two
isoforms of a poly(C) binding protein (PCBP, also known as  CP or
hnRNPE),15-20 and expression of an isoform
PCBP1 is induced by hypoxia in PC12 cells.15
HIPBS sequence is analogous to a pyrimidine-rich sequence located
within the EPO RNA-protein-binding region.9 Both sequences
are pyrimidine-rich and contain short stretches of cytidines
interrupted by one or two uridines.13 In view of these
sequence similarities between the TH and the EPO mRNA, we hypothesized
that the same PCBP protein should bind to the pyrimidine-rich
HIPBS-like element of the EPO mRNA. Indeed, we determined that two
isoforms of PCBP are present in the ribonucleoprotein complex
associated with the EPO mRNA 3' UTR. Expression of PCBP isoforms,
however, is not induced by hypoxia; nor is formation of the
ribonucleoprotein complex associated with pyrimidine-rich sequence in
the EPO 3' UTR.
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MATERIALS AND METHODS |
Cell culture and preparation of cytoplasmic extracts.
PC12, Hep3B, and HepG2 cells were grown in Dulbecco's modified
Eagle's medium (DMEM)/F12 medium containing 10% fetal bovine serum,
100 units/mL penicillin, and 100 µg/mL streptomycin, as described
previously.12-14 Cells reaching 75% to 85% confluence were exposed to either normoxia or hypoxia (1% O2) in an
oxygen-regulated incubator (Forma Scientific Marietta, OH)
for 24 hours.12-14 Cytosolic protein extracts were obtained
as described previously. 12-14
Plasmid constructs and RNA transcript.
A 210 bp StuI-BglII fragment of human EPO complementary
DNA (cDNA) (a gift from Dr H.F. Bunn, Fig
1A) corresponding to the last 22 bases of the coding region and the
first 188 bases of the 3' UTR, which contains the HIPBS-like
site, was subcloned into SmaI-BglII sites of
transcription vector pSP73. Templates for transcription were obtained
by linearizing pSP73-EPO vector with either DdeI, NcoI,
or BglII (Fig 1A). The 93-base-long transcript, obtained from
the template linearized with DdeI, included only the
pyrimidine-rich stretch. Comparison of this sequence in different species, starting at the translation stop codon, is shown in Fig 1B. In
vitro transcriptions were performed using T7 RNA polymerase in the
presence of 50 µCi of [-32P] uridine triphosphate
(UTP) (3000 Ci/mmol), 2.5 mmol/L of all four unlabeled nucleotides, 20 mmol/L dithiothreitol (DTT), and RNasin at 42°C for 60 minutes, as
described previously.12 TH transcripts were prepared as
described previously.12-14 The sequences of the synthetic
wild-type and mutated EPO oligoribonucleotides are shown in Fig 1C.
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| Fig 1.
(A) Schematic representation of human EPO cDNA, and
location of restriction sites DdeI, NcoI, and
BglII within the 3' UTR used to generate EPO transcripts.
The hatched area represents the HIPBS-like region. TGA - translation
stop codon. Bracket marks the cDNA used to generate the riboprobe. (B)
Alignment of pyrimidine-rich tracts in the 3' UTR of EPO mRNA
from different species. Sequences start with the translation stop
codon. The first pyrimidine-rich tract conserved in primates is
indicated by italics and underlined. The second motif conserved in
various species is indicated by bold type and underlined. Gene bank
accession numbers for each EPO mRNA are X02157 (human), M18189
(monkey), L10608 (rat), M12482 (mouse), L10607 (swine), U44762
(bovine). (C) Sequences of wild-type TH and EPO HIPBS elements, and
localization of mutations (MUT). The G residue within the protein
binding site is underlined.
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RNA-protein binding reactions and electrophoresis of complexes.
Gel shift reactions and UV light cross-linking electrophoresis of
RNA-protein complexes were performed essentially as described previously.12-14 Briefly, cytoplasmic protein extracts (20 to 40 µg) were incubated in the binding buffer (10 mmol/L HEPES, pH 7.9, 5 mmol/L MgCl2, 50 mmol/L KCl, and 10% glycerol, 200 ng/ml Escherichia coli transfer RNA [tRNA], and 1 mmol/L DTT)
at room temperature for 15 minutes. Labeled RNA transcripts or
oligoribonucleotides (100,000 cpm/reaction) were added, and the
reaction mixture was incubated for 10 minutes. After completion of the
binding reaction, RNase T1 (10 units) and heparin sulfate (final
concentration 2.5 mg/mL) were added sequentially to the reaction
mixture for 5 minutes each. In competition experiments the cold
competitor probe was added before the radioactive probe. For UV light
cross-linking, binding reactions were exposed to 1 × 106 J/cm2. The cross-linked
reaction was separated on 9% acrylamide gel under reducing conditions
(100 mmol/L DTT in the sample buffer). When TH and EPO RNA-protein
interactions were compared in the same experiment four times less of
the PC12 protein extract was used with the TH probe to obtain
autoradiographically comparable signal.
For electroelution of protein factors, the binding reactions were
performed using 10 µg of unlabeled EPO DdeI transcript and 1.5 to 2 mg of protein extract from HepG2 cells in a 300 µL reaction volume. The control reactions were identical except for the RNA. Complexes were visualized by corunning one reaction that contained 32P-labeled RNA. Identified complexes were excised,
macerated, and loaded into the elution cell in the inside cup buffer,
which contained 5 mmol/L Tris-acetate, pH 8, 0.1% SDS, 0.1 mmol/L
EDTA. Proteins were eluted overnight at 4°C with a constant voltage
of 100 mV in the outside cup buffer (50 mmol/L Tris-acetate, pH 8, 0.1% SDS, 0.1 mmol/L EDTA). The inside and outside compartments were separated by dialysis membrane with molecular weight cut-off
(MWCO) 12,000 to 14,000 (Spectra/por*2, Spectrum Medical
Inc). The eluted proteins were concentrated using
Microcon-10 microconcentrators, resuspended in the sample buffer, and
subjected to 9% SDS-PAGE analysis. Proteins then were transferred onto
the nitrocellulose with a semidry blotter (Bio-Rad, Hercules,
CA). The blots were blocked in Tris-buffered saline, Tveen
20 (TBST) with 5% nonfat milk, and were first probed with
a specific anti-PCBP1 antibody,15 and then
stripped, and reprobed with a specific anti-PCBP2 antibody (gift from A.V. Gamarnik and R. Andino) in TBST with 5% dry milk. The
signal was visualized by exposing blots to chemiluminescence reagents
(Amersham, Arlington Heights, IL).
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RESULTS |
Similarities in the formation of ribonucleoprotein complex associated
with EPO and TH HIPBS-like elements.
Analysis of the 3' UTR of the EPO mRNA showed sequences highly
homologous to the HIPBS element in TH mRNA (Fig 1B). The 3' UTR
of primate EPO mRNA contains two repeats of cytidine/uridine-rich motifs, separated by a sequence richer in purines. The first motif is
present only in the 3' UTR of primates (Fig 1B, underlined and
italicized sequences), whereas the second motif is conserved in various
species (Fig 1B, underlined bold sequences).
Because of these similarities in the sequence we hypothesized that the
pyrimidine-rich motifs in EPO and TH mRNA bind the same protein
factors. Binding reactions were performed using EPO or TH transcripts
containing HIPBS-like sequences and cytoplasmic protein extracts from
Hep3B, HepG2, or PC12 cells (Fig 2). The two EPO transcripts (BglII and DdeI) formed essentially
the same two complexes with protein extracts from either Hep3B or HepG2 cells (Fig 2A, lanes 3 to 8). This indicates that the protein-binding sites are restricted within the 93 bases (bases 738 to 860) of the EPO
DdeI transcript. The faster-migrating complex (solid arrow) migrated with the same mobility as the complex formed by TH transcript with protein factors from PC12 cells (Fig 2A, lane 2) or the complex formed by EPO oligoribonucleotide with protein factors from Hep3B or
HepG2 cells (Fig 2A, lanes 10 and 11). This EPO oligoribonucleotide corresponds to the pyrimidine/cytidine-rich sequence (HIPBS-like motif)
in EPO mRNA conserved in various species. A second,
slower-migrating complex (open arrow) was usually visible in the
binding reactions with both EPO transcripts, but not with the EPO
HIPBS-like oligoribonucleotide. These data indicate that the
faster-migrating ribonucleoprotein complex is associated with the
conserved 818 to 830 base fragment of EPO 3' UTR, which contains
the HIPBS-like motif, whereas the slower-migrating complex is most
likely associated with the less conserved more proximal region
corresponding to bases 738 to 818 of the EPO 3' UTR.
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| Fig 2.
EPO transcripts containing HIPBS-like sequence form
ribonucleoprotein complexes with protein extracts from Hep3B and HepG2
that migrate with the same mobility as the TH mRNA-associated complex.
(A) RNA gel-shift assay with TH transcript (TH-tr, lanes 1 to 2) and
PC12 cells extract (10 µg), EPO BglII transcript (EPO
BglII-tr, lanes 3 to 5), EPO DdeI
transcript (EPO DdeI-tr, lanes 6 to 8), and EPO
oligoribonucleotide (EPO-HIPBS, lanes 9 to 11) and with proteins (40 µg) from Hep3B (H3B) and HepG2 (HG2) cells. Solid arrow indicates the
main, faster-migrating complex. Open arrow indicates the second,
slower-migrating complex, visible only when EPO transcripts are used.
FP - free probe. Free probes migrate only in lanes 1, 3, 6, and 9. Note
that less PC12 protein extract was used in the binding reaction with
the TH transcript to obtain autoradiographically comparable signal
between TH and EPO complexes. (B) Competition of the complexes formed
by EPO DdeI transcript with proteins (40 µg) from Hep3B
(lanes 1 to 4) or Hep2G (lanes 5 to 8) by TH HIPBS (lanes 2 and 6) or
poly(C) RNA (lanes 3 and 7) but not by poly(U) RNA (lanes 4 and 8).
Solid arrow indicates specific complex. Open arrow indicates
slower-migrating complex.
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To determine the specificity of the binding, competition reactions were
performed (Fig 2B). Formation of the complex between EPO DdeI
transcript and cytoplasmic protein extracts from either Hep3B or HepG2
cells, which migrated with the same mobility as the TH mRNA-protein
complex (solid arrow), was abolished when TH HIPBS oligoribonucleotide
or poly(C) RNA was added to the binding reaction, but not when poly(U)
RNA was added (Fig 2B). On the other hand, formation of the slower
migrating complex (open arrow) was resistant to the competition with TH
HIPBS, although poly(C) RNA also competed with it. These results
indicate that the faster-migrating complex associated with EPO
DdeI transcript has an affinity for the same poly(C)-binding
protein factors as TH HIPBS, whereas the slower-migrating complex
involves other protein(s) specific to the EPO transcript.
To further identify EPO RNA-protein complexes, binding reactions
containing TH probe or EPO NcoI or DdeI transcripts and
cytoplasmic protein extracts from either PC12, Hep3B, or HepG2 cells
were UV light cross-linked and analyzed by SDS-PAGE (Fig 3). The
binding reactions were performed in the presence or absence of poly(U) RNA as a nonspecific competitor. In the absence of poly(U), TH transcript formed a 50 kD complex and additional complexes migrating between 50 and 80 kD (lane 1) with proteins from PC12 cells. In the
presence of poly(U), TH transcript formed only one complex migrating at
50 kD (lane 2), as we described previously.15 In the
absence of poly(U), both EPO transcripts formed two complexes with
protein factors from PC12 cell line (lanes 3 and 5) migrating at
approximately 50 and 60 kD. Addition of poly(U) RNA, which does not
affect formation of the complexes visible in the gel retardation assays
(Fig 2B), completely abolished formation of the 60 kD complex with EPO
transcripts, leaving only the 50 kD complex (lanes 4 and 6).
Importantly, formation of the 50 kD complex with EPO DdeI
transcript was specifically abolished by addition of poly(C) RNA or TH
HIPBS oligoribonucleotide (lanes 7 to 10). This indicates that the same
complex is formed with protein extracts by both TH and EPO DdeI
transcripts. The same 50 kD complex was formed with the EPO
DdeI transcript in the presence of poly(U) RNA when protein
extracts were used from either Hep3B or HepG2 cells
(Fig 3B). In the case of protein extracts
from both Hep3B and HepG2 cells, an additional, fainter band was
visible at 70 kD. The presence of this band was less consistent as
compared with the 50 kD complex.
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| Fig 3.
SDS-PAGE analysis of the UV light cross-linked
RNA-protein complexes. (A) Characterization of the complexes formed by
TH transcript (TH-tr), and by two EPO transcripts (NcoI-tr and
DdeI-tr) and proteins (40 µg) from PC12 cells. Poly(U) RNA,
TH HIPBS, or poly(C) RNA were added to the binding reaction as
competitors (+ 50 ng, ++ 100 ng). Solid arrow points to the 50 kD
complex. FP - free probe. (B) Comparison of complexes formed with the
EPO DdeI transcript and protein factors from PC12 (lane 2),
Hep3B (H3B, lane 3), and HepG2 (HG2, lane 4) before (lanes 2, 4, and 6)
and after (lanes 3, 5, and 7) addition of 100 ng of poly(U) RNA. Lane 1 - free DdeI EPO probe. Solid arrow indicates the 50 kD complex.
Open arrow indicates the 70 kD complex formed with extracts from Hep3B
and HepG2 cells.
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Because the EPO-HIPBS is the binding site for the protein factors, we
attempted to characterize binding of protein factors to this fragment
of EPO RNA (Fig 4). EPO HIPBS formed a
single complex identified in the RNA gel shift assay (Fig 4A, lane 1); formation of this complex was abolished by adding either TH or EPO
HIPBS oligoribonucleotides, EPO DdeI transcript, or poly(C) RNA
(Fig 4A, lanes 2 to 5). This complex, identified in a gel-shift assay,
corresponded to a single 50 kD complex identified after UV light
cross-linking of the binding reaction and SDS-PAGE analysis of the
complexes (Fig 4B, lane 1). Again, formation of this complex was
abolished by either poly(C) or TH HIPBS RNA (Fig 4B, lanes 2 and 3).
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| Fig 4.
Formation of the ribonucleoprotein complex associated
with EPO HIPBS-like oligoribonucleotide. (A) Gel-shift analysis of the
binding reaction of EPO oligoribonucleotide and protein factors from
HepG2 cells. EPO oligoribonucleotide is sufficient to form a complex
with proteins from HepG2 cells (lane 1). Formation of this complex is
prevented by competition with 100 ng of cold EPO (lane 2) or TH HIPBS
(lane 3) oligoribonucleotides, EPO DdeI transcript (lane 4), or
poly(C) RNA (lane 5). (B) Analysis of the EPO
oligoribonucleotide-protein complex after UV light cross-linking and
SDS-PAGE. EPO oligoribonucleotide forms a 50 to 55 kD complex with
cytoplasmic proteins from HepG2 cells (lane 1). Formation of this
complex is abolished by competition with 100 ng of poly(C) RNA (lane 2)
or TH HIPBS (lane 3). (C) Gel-shift analysis of complexes formed by
proteins from HepG2 cells and wild-type (wt) or mutated (M-1, 2, 3, 4)
EPO HIPBS. Bracket indicates the specific complex. (D) Gel-shift
analysis of the HepG2 protein complex associated with EPO HIPBS in the
presence of increasing concentrations of RT1 (lanes 1 to 4). Bracket
indicates the specific complex. FP-freeprobe
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The predicted protein-binding site within the EPO HIPBS is the
cytidine-rich motif CCCUCCCCCGCC (Fig 1B). Synthetic
oligoribonucleotides with mutations within this predicted
protein-binding motif were used in the binding reactions (Fig 4C).
Mutations within the EPO that affected this cytidine-rich motif
abolished protein binding (Fig 4C, lanes 2 to 4). Mutations outside
this motif allowed for some decreased residual binding (Fig 4C, lane
5). This indicates that the predicted cytidine-rich motif is the
protein-binding site. Because of the guanidine residue within the
cytidine-rich region, formation of the complex was tested for
sensitivity to the RNAse T1 (Fig 4D). Clearly, formation of this
complex was abolished by increasing concentrations of RNase T1 (RT1,
Fig 4D). This proves, as predicted, that the G residue in this EPO
HIPBS-like element is located in the protein-binding site and to some
extent is protected from RT1 activity by the protein binding. The
higher doses of RT1, however, overcame the protective effects of bound proteins.
The ribonucleoprotein complex associated with EPO HIPBS-like RNA
contains 40 kD PCBP.
The protein factor binding to the TH HIPBS in a hypoxia-inducible
manner is the 40 kD PCBP.15 To determine whether PCBP participates in formation of the complex associated with the EPO HIPBS-like element, binding reactions were performed using EPO DdeI transcript and proteins from HepG2 cells. Gel-shift assays were performed, and protein factors were electroeluted from a complex
identified as migrating with the same mobility as the TH
HIPBS-associated complex (lower complex). Proteins were also eluted
from the EPO mRNA-protein complexes that migrated with lower mobility
(upper complex) than the TH-protein complexes, and from gel slices
containing binding reactions without RNA. The eluted proteins were
analyzed by Western blot with specific anti-PCBP1 or
PCBP2 antibodies (Fig 5). The
lower EPO mRNA protein complex specifically contained both isoforms of
PCBP (Fig 5, lane 5). This protein was absent in the control binding
reactions performed without RNA (Fig 4, lane 4), and in the upper
complex and its control (lanes 2 and 3). Note that PCBP2
migrates slightly slower, most likely because of the posttranslational
modifications.
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| Fig 5.
Identification of PCBP as part of the ribonucleoprotein
complex associated with EPO HIPBS-like element in HepG2 cells Proteins
electroeluted from the EPO DdeI transcript-associated complexes
were analyzed by Western blot technique. The same membrane was first
probed with specific anti-PCBP1 antibody,and then stripped
and reprobed with anti-PCBP2 antibody. The lower complex
(corresponding to the TH-HIPBS-associated complex) contains both
isoforms of PCBP (lane 5), which is absent in the eluates from gel
slices containing binding reaction without RNA (lane 4) or in the upper
complex (lanes 2 and 3). Lane 1, 100 µg of HepG2 protein extract.
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To determine whether binding of protein factors to the HIPBS-like
element within the 3' UTR of EPO mRNA is regulated by hypoxia, binding reactions were performed with protein extracts from PC12, Hep3B, and HepG2 cells exposed to 21% or 1% O2 and EPO
BglII, DdeI transcripts, EPO HIPBS oligoribonucleotide,
or TH transcript and analyzed by gel-shift assay of UV light
cross-linking and SDS-PAGE. Clearly, hypoxia did not induce formation
of the complexes associated with any of the EPO or TH probes and
protein extracts from either Hep3B or HeG2 cells
(Fig 6A, 6B, lanes 1 to 4, 6C, lanes 4 to
7). However, hypoxia induced formation of the complexes with either EPO
DdeI (Fig 6B, lanes 6 to 7) or TH (Fig 6C, lanes 2 to 3)
transcripts when protein extracts were used from PC12 cells. In
contrast to PC12 cells,15 in HepG2 or Hep3B cells hypoxia
did not induce expression of the PCBP (not shown).
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| Fig 6.
Formation of the ribonucleoprotein complex associated
with EPO mRNA is not hypoxia-inducible. (A) Gel-shift
analysis of the protein complexes associated with EPO
BglII (lanes 1 to 5), EPO DdeI (lanes 6 to 10)
transcripts or EPO HIPBS (lanes 11 to 15) and protein extracts (40 µg) from either Hep3B or HepG2 cells during normoxia (21%
O2) and hypoxia (1% O2). (B) SDS-PAGE analysis
of UV light cross-linked complexes formed by EPO DdeI-transcript and
protein factors (40 µg) from Hep3B (lanes 1, 2), HepG2 (lanes 3, 4),
and PC12 cells (lanes 5, 6) that were exposed to normoxia (21%
O2) or hypoxia (1% O2). (C) SDS-PAGE analysis
of UV light cross-linked complexes formed by TH transcript and protein
factors from PC12 (10 µg, lanes 2, 3), Hep3B (40 µg, lanes 4, 5),
and HepG2 cells (40 µg, lanes 6, 7) that were exposed to normoxia
(21% O2) or hypoxia (1% O2). FP - free
probe.
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DISCUSSION |
In this study we identified two isoforms of a 40 kD PCBP17
as a part of a ribonucleoprotein complex associated with a
cytidine-rich sequence located within the 3' UTR of EPO mRNA.
This is the first identification of protein factors that are associated
with EPO mRNA. The conserved cytidine-rich sequence is located within
the putative EPO mRNA stability region. Direct evidence regarding the
role of this region in regulation of EPO mRNA stability is still
missing. However, deletion of the EPO 3' UTR downstream from the
pyrimidine-rich region results in stabilization of the EPO
mRNA.7 In contrast, deletion of the 100 bases including the
pyrimidine-rich region results in destabilization of the mRNA, although
under conditions of prolonged EPO mRNA half-life due to the use of
actinomycin D.8 In addition, some other mRNA, such as
2 globin,16-19 collagen
1(I),20 and TH15 mRNA, were shown to use
cytidine-rich motifs as determinants of their stability and to bind the
PCBP. Thus, it is likely that the cytidine-rich motif represents the
EPO mRNA stabilizing element and that PCBP plays a stabilizing role in
maintaining constitutive stability of EPO mRNA.
The role of this cytidine-rich motif in potential hypoxic regulation of
EPO mRNA stability is less clear. On the basis of functional studies,
this regulatory stability element in the 3' UTR of TH mRNA is
necessary but not sufficient for the hypoxic regulation of TH mRNA
stability.15 The same may be true in the case of EPO mRNA.
In contrast to TH mRNA, however, formation of the ribonucleoprotein
complexes associated with the HIPBS-like element is not inducible by
hypoxia in the EPO-synthesizing cell lines such as Hep3B or HepG2. This
finding is consistent with inability to induce expression of PCBP by
hypoxia in HepG2 or Hep3B cells, and with the previously reported lack
of hypoxic inducibility of erythropoietin RNA binding proteins (ERBP)
binding to EPO mRNA.9 This may indicate that if EPO mRNA
stability is regulated by hypoxia, protein factors other than the PCBP
may be involved in this regulation. In that respect, several studies have implicated that hypoxia-inducible protein binding to the AU-rich
instability elements within the 3' UTR of the vascular endothelial growth factor mRNA may regulate stability of this mRNA
during hypoxia.21,22 In this respect the 3' UTR of
EPO mRNA may contain instability elements because deletion of the conserved distal part of EPO 3' UTR results in stabilization of the remaining EPO mRNA.8 In addition, a single instability element, an AUUUA motif (bases 1100 to 1105), is
present in the distal part of EPO 3' UTR. Thus, perhaps the role
of the EPO mRNA instability elements located downstream from the EPO
mRNA stability element should be examined in hypoxic regulation of EPO mRNA.
We have clearly showed that there are tissue-specific differences in
regulation of PCBP expression by hypoxia. In PC12 cells expression of
PCBP1 but not PCBP2 is induced by
hypoxia.15 In contrast, neither of the PCBP is induced by
hypoxia in Hep3B or HepG2 cells. This finding is not surprising
because, in addition to universal mechanisms exist specific mechanisms
for oxygen-sensing and regulating gene expression. For example,
regulation of TH gene expression by reduced oxygen tension is tissue
specific for carotid body and PC12 cells but not for other
catecholaminergic cells.23 In addition, oxygen-sensing
mechanisms in PC12 cells or carotid body cells are more sensitive than
in other tissues. In this respect, TH gene expression is induced by
even mild (5%) hypoxia, whereas induction of EPO requires stronger
reduction in oxygen tension, usually to 1%.24 Finally,
different molecular mechanisms are involved in transcriptional
regulation of TH and of EPO gene expression by hypoxia. Induction of
EPO gene transcription involves predominantly Hif-1,25
whereas induction of TH gene transcription involves binding of c-fos
and jun-b to the Ap1 site on the TH promoter.26
Analysis of the UV light cross-linked complexes showed that the main
specific complex associated with EPO DdeI transcript in
different cell lines is 50 kD, which corresponds to the binding protein
PCBP of 40 kD. The presence of this 50 kD complex was not reported
previously. The previous studies identified the EPO mRNA-associated
complexes as 70 and 135 kD (ERBP).9 In our experiments we
have inconsistently observed formation of a 70 kD complex with proteins
from Hep3B and HepG2 cells. It is possible that this 70 kD complex
corresponds with the one reported previously by others.9 We
have not identified the 135 kD complex, however. One possible reason
for this discrepancy is that the 135 kD complex may contain more than
one protein factor because analysis of the EPO RNA-protein complexes in
that study was performed in the absence of reducing agents in the
sample buffer.9 In our experiments the cross-linked
reactions are analyzed in the presence of 100 mmol/L DTT, that is,
under reducing conditions. In this study we did not attempt to
identify other protein factors binding to the EPO mRNA, that may be
involved in formation of the slower-migrating complex identified in the
gel shift assays. It is possible that this complex corresponds to the
70 kD complex identified by SDS-PAGE .
In this study we did not determine whether the PCBP are the only
proteins present in the ribonucleoprotein complex associated with the
EPO mRNA pyrimidine-rich sequence or whether other proteins also
interact with PCBP. Recently it was proposed that ERBP interacts with
the heat-shock protein, hsp70.27 During normoxia, ERBP is
bound to the hsp 70, and its binding to the EPO mRNA is low. During
hypoxia, hsp 70 is sequestered; this allows the ERBP to bind fully to
the EPO mRNA.27 Further studies are required to identify
such potential interactions.
Identification of the PCBP protein in the complex associated with EPO
mRNA stability represents a significant step in understanding posttranscriptional regulation of EPO gene expression.
| |
ACKNOWLEDGMENT |
We thank A. Gamarnik and R. Andino for anti-PCBP2 antibody,
H.F. Bunn for EPO cDNA, and G. Doerman for preparation of the figures.
| |
FOOTNOTES |
Supported by NIH grants no. NIH HL51078, HL58687, and American Heart
Association Grant-in-Aid 9750110N.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address correspondence to Maria F. Czyzyk-Krzeska, Department of
Molecular and Cellular Physiology, University of Cincinnati, College of
Medicine, PO Box 670576, Cincinnati, OH 45267-0576; e-mail:
Maria.Czyzykkrzeska{at}uc.edu.
| |
REFERENCES |
1.
Jelkmann W:
Erythropoietin: Structure, control of production, and function.
Physiol Rev
72:449, 1992[Free Full Text]
2.
Eckardt KU, Koury ST, Tan CC, Schuster SJ, Kaissling B, Ratcliffe PJ, Kurtz A:
Distribution of erythropoietin producing cells in rat kidneys during hypoxic hypoxia.
Kidney Int
43:815, 1993[Medline]
[Order article via Infotrieve]
3.
Goldberg MA, Glass GA, Cunningham JM, Bunn HF:
The regulated expression of erythropoietin by two human hepatoma cell lines.
Proc Natl Acad Sci USA
84:7972, 1987[Abstract/Free Full Text]
4.
Bunn HF, Poyton RO:
Oxygen sensing and molecular adaptation to hypoxia.
Physiol Rev
76:839, 1996[Abstract/Free Full Text]
5.
Goldberg MA, Gout CC, Bunn HF:
Erythropoietin mRNA levels are governed by both the rate of gene transcription and posttranscriptional events.
Blood
77:271, 1991[Abstract/Free Full Text]
6.
Bunn HF, Poyton RO:
Oxygen sensing and molecular adaptation to hypoxia.
Physiol Rev
76:839, 1996
7.
Ho V, Acquaviva A, Duh E, Bunn HF:
Use of a marked erythropoietin gene for investigation of its cis-acting elements.
J Biol Chem
270:10084, 1995[Abstract/Free Full Text]
8.
McGary EC, Rondon IJ, Beckman BS:
Post-transcriptional regulation of erythropoietin mRNA stability by erythropoietin mRNA-binding protein.
J Biol Chem
272:8628, 1997[Abstract/Free Full Text]
9.
Rondon IJ, MacMillan LA, Beckman BS, Goldberg MA, Schneider T, Bunn HF, Malter JS:
Hypoxia up-regulates the activity of a novel erythropoietin mRNA binding protein.
J Biol Chem
266:16594, 1991[Abstract/Free Full Text]
10.
Rondon IJ, Scadurro AB, Wilson RB, Beckman BS:
Changes in redox affect the activity of erythropoietin RNA binding protein.
FEBS Lett
359:267, 1995[Medline]
[Order article via Infotrieve]
11.
Czyzyk-Krzeska MF, Furnuri BA, Lawson EE, Millhorn DE:
Hypoxia increases rate of transcription and stability of tyrosine hydroxylase mRNA in pheochromocytoma (PC12) cells.
J Biol Chem
269:760, 1994[Abstract/Free Full Text]
12.
Czyzyk-Krzeska MF, Dominski Z, Kole R, Millhorn DE:
Hypoxia stimulates binding of a cytoplasmic protein to a pyrimidine rich sequence in the 3' untranslated region of rat tyrosine hydroxylase mRNA.
J Biol Chem
269:9940, 1994[Abstract/Free Full Text]
13.
Czyzyk-Krzeska MF, Beresh JE:
Characterization of the hypoxia inducible protein binding site within the pyrimidine rich tract in the 3' untranslated region of the tyrosine hydroxylase mRNA.
J Biol Chem
271:3293, 1996[Abstract/Free Full Text]
14.
Czyzyk-Krzeska MF, Paulding WR, Beresh JE, Kroll SL:
Post-transcriptional regulation of tyrosine hydroxylase gene expression by oxygen in PC12 cells.
Kidney Int
51:585-590, 1997[Medline]
[Order article via Infotrieve]
15.
Paulding WR, Czyzyk-Krzeska MF:
Regulation of tyrosine hydroxylase mRNA stability by protein-binding, pyrimidine-rich sequence in the 3' untranslated region.
J Biol Chem
274:2532, 1999[Abstract/Free Full Text]
16.
Wang X, Kiledjian M, Weiss IM, Liebhaber SA:
Detection and characterization of a 3' untranslated region ribonucleoprotein complex associated with human -globin mRNA stability.
Mol Cell Biol
15:1769, 1995[Abstract]
17.
Kiledjian M, Wang X, Liebhaber SA:
Identification of two KH domain proteins in the -globin mRNP stability complex.
EMBO J
14:4357, 1995[Medline]
[Order article via Infotrieve]
18.
Weiss IM, Liebhaber SA:
Erythroid cell-specific mRNA stability elements in the 2-globin 3' nontranslated region.
Mol Cell Biol
15:2457, 1995[Abstract]
19.
Holcik M, Liebhaber SA:
Four highly stable eukaryotic mRNAs assemble 3' untranslated region RNA-protein complexes sharing cis and trans components.
Proc Natl Acad Sci USA
94:2410, 1997[Abstract/Free Full Text]
20.
Stefanovic B, Hellerbrand C, Holcik M, Briendl M, Liebhaber SA, Brenner DA:
Posttranscriptional regulation of collagen a1(I) mRNA in hepatic stellate cells.
Mol Cell Biol
17:5201, 1997[Abstract]
21.
Levy AP, Levy NS, Goldberg MA:
Posttranscriptional regulation of vascular endothelial growth factor by hypoxia.
J Biol Chem
271:2746, 1996[Abstract/Free Full Text]
22.
Levy AP, Levy NS, Goldberg MA:
Hypoxia-inducible protein binding to vascular endothelial growth factor mRNA and its modulation by the von Hippel-Lindau protein.
J Biol Chem
271:25492, 1996[Abstract/Free Full Text]
23.
Czyzyk-Krzeska MF, Bayliss DA, Lawson EE, Millhorn DE:
Regulation of tyrosine hydroxylase gene expression in the rat carotid body by hypoxia.
J Neurochem
58:1538, 1992[Medline]
[Order article via Infotrieve]
24.
Czyzyk-Krzeska MF:
Molecular aspects of oxygen sensing in physiological adaptation to hypoxia.
Respir Physiol
110:99, 1997[Medline]
[Order article via Infotrieve]
25.
Wang GL, Semenza GL:
Characterization of hypoxia-inducible factor 1 and regulation of DNA binding activity by hypoxia.
J Biol Chem
268:21513, 1993[Abstract/Free Full Text]
26.
Norris ML, Millhorn DE:
Hypoxia-induced protein binding to O2-responsive sequences on the tyrosine hydroxylase gene.
J Biol Chem
270:23774, 1995[Abstract/Free Full Text]
27.
Scandurro AB, Rondon IJ, Wilson RB, Tennenbaum SA, Garry RF, Beckman BS:
Interaction of erythropoietin RNA binding protein with erythropoietin RNA requires an association with heat shock protein 70.
Kidney Int
51:579, 1997[Medline]
[Order article via Infotrieve]
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ABSTRACT
INTRODUCTION