From the Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0576
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ABSTRACT |
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The stability of mRNA for tyrosine
hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis,
is regulated by oxygen tension in the pheochromocytoma-derived PC12
cell line. We previously identified a pyrimidine-rich 27-base-long
protein-binding sequence in the 3'-untranslated region of TH mRNA
that is associated with hypoxia-inducible formation of a
ribonucleoprotein complex (hypoxia-inducible protein-binding site
(HIPBS)). In this study, we show that HIPBS is an mRNA stabilizing
element necessary for both constitutive and hypoxia-regulated stability
of TH mRNA. The mutations within this sequence that abolish protein
binding markedly decrease constitutive TH mRNA stability and ablate
its hypoxic regulation. A short fragment of TH mRNA that contains the wild-type HIPBS confers the increased mRNA stability to the reporter chloramphenicol acetyltransferase mRNA. However, it is not
sufficient to confer hypoxic regulation. The HIPBS element binds two
isoforms of a 40-kDa poly(C)-binding protein (PCBP). Hypoxia induces
expression of the isoform 1, PCBP1, but not the isoform 2, PCBP2, in PC12 cells.
Tyrosine hydroxylase
(TH),1 the rate-limiting
enzyme in the biosynthesis of catecholamines, is expressed in specific
populations of neurons in the central and peripheral nervous systems,
in the neuroendocrine cells of the adrenal medulla and carotid body, and in cultured cell lines such as the pheochromocytoma-derived PC12
cell line. Regulation of TH gene expression at the level of gene
transcription is well documented. Recently, there has been growing
evidence that TH mRNA is also regulated at the level of mRNA
stability. TH mRNA is a stable message with a half-life that varies
from 9 to 16 h in various subclones of PC12 cells (1-3). It is
enhanced during differentiation of neuroblastoma cells (1) and during
stimulation of the protein kinase C pathway in PC12 cells (2). In
contrast, the stability of TH mRNA does not change in PC12 cells
during stimulation of TH mRNA expression by dexamethasone or
forskolin (3). A recent study demonstrated substantial differences in
basal TH mRNA turnover rates between different neuronal populations
from as short a time as 6-7 h, in the dopaminergic neurons of the
arcuate nucleus, to as long as 11-23 h, in the dopaminergic
midhypothalamic neurons (4). In addition, TH mRNA is destabilized
in the dopaminergic cells of the arcuate nucleus in a manner that
corresponds to the rhythmic output displayed by these neurons (4).
Our laboratory demonstrated that hypoxia augments the stability of TH
mRNA in PC12 cells (5). We identified a 27-base-long pyrimidine-rich sequence within the TH mRNA 3'-untranslated region (UTR) (1552-1578 bases of TH mRNA) that binds protein factors in a
hypoxia-inducible manner (hypoxia-inducible protein-binding sequence
(HIPBS)) in PC12 cells (6, 7), catecholaminergic cells of the superior
cervical ganglia, and the dopaminergic cells of the carotid body (8).
Mutational analysis revealed that the optimal protein-binding site is
represented by the motif (U/C)(C/U)CCCU within the
pyrimidine-rich sequence, where the underlined cytidines are the core
binding site (7). This motif is conserved in TH mRNAs from
different species. These results imply that formation of the
ribonucleoprotein complex associated with HIPBS may be involved in
physiological regulation of TH mRNA stability in catecholaminergic cells. Thus, identification of the role of this element in the regulation of TH mRNA stability is critically important to further investigations in this area.
In the present study, we investigated the role of HIPBS in regulating
the stability of TH mRNA during normoxia and hypoxia. We show that
HIPBS is a stabilizing element necessary for maintaining constitutive
and hypoxia-regulated TH mRNA half-life. We also show that the
HIPBS-binding protein is represented by two isoforms of a 40-kDa
poly(C)-binding protein (PCBP, also known as Materials--
All chemicals were purchased from either Fisher
or Sigma and enzymes from Promega or Life Technologies, Inc.
Cell Culture--
PC12 cells were grown in Dulbecco's modified
Eagle's medium/F-12 supplemented with 15 mM HEPES buffer,
10% fetal bovine serum, 100 units/ml penicillin, and 100 mg/ml
streptomycin, as described previously (5, 6).
Plasmid Constructs--
The 5'-marked TH cDNA constructs
were obtained by inserting full-length TH cDNA (see Fig.
1A), which had either a wild-type or mutated protein-binding
site, into the BamHI site of pcDNA3 expression vector
(Invitrogen). Expression of TH mRNA from these vectors results in
exogenous TH transcripts containing 25 bases of the TH 5'-UTR region
and an additional 26-base-long sequence (BamHI-HindIII) upstream from the
plasmid DNA (see Fig. 1A).
The chimeric CAT-TH constructs were generated by inserting the
KpnI-ApaI fragment of TH 3'-UTR that contains
either a wild-type or mutated protein-binding site into the respective
restriction sites behind the chloramphenicol acetyltransferase (CAT)
gene within the pcDNA3CAT vector (see Fig. 3A). The
derived chimeric mRNAs are referred to, respectively, as
CAT-wtTH (wild-type) or CAT-mutTH (mutant).
Stable Transfections--
Transfections were performed using
LipofectAMINE (Life Technologies Inc.), according to the
manufacturer's protocol. Briefly, 10 µg of plasmid DNA and 10 µl
of LipofectAMINE were combined in a polystyrene tube containing 800 µl of Dulbecco's modified Eagle's medium/F-12 without serum or
antibiotic. This mixture was added to 60% confluent PC12 cells growing
on a 100-mm2 dish. After 5-7 h, fetal bovine serum and
antibiotics were added to achieve a concentration identical to that
used for maintaining the cells. The cells were allowed to recover for
72 h and were passaged 1:4 into a selective medium containing 400 µg/ml G418 (Life Technologies Inc.). After 8-12 weeks, the colonies
were pooled together and used for analysis. Each pool of stably
transfected cells contained 20-100 individual colonies.
Ribonuclease Protection Assay (RPA)--
RNA from transfected
cells was extracted with TRI reagent (MRC, Inc., Cincinnati, OH) and
was digested with RNase-free DNase (Ambion),
phenol-chloroform-extracted, and precipitated. The marked TH antisense
probe was transcribed from the SP73 transcription vector (Promega)
containing the HindIII-PstI fragment of the
5'-region of marked TH cDNA (see Fig. 1A) using SP6
polymerase. The CAT (see Fig. 3A) and 18 S antisense
riboprobes were transcribed from TRI-CAT or T7-18 S templates (Ambion)
using T7 polymerase. TH and CAT probes were labeled to the high
specific activity using [ RNA·Protein-binding Reactions (6, 7)--
Forty micrograms of
the S-100 fraction of cytosolic proteins and the
[ Poly(C)-Agarose Affinity Purification--
This procedure was
performed essentially as described previously (9). The S-100 protein
fraction (15-20 mg of protein extract) from PC12 cells was treated
with micrococcal nuclease (500 units; Worthington) in the presence of 1 mM CaCl2 for 1 h at room temperature. The
reaction was stopped by adding 5 mM EGTA, and extracts were incubated for 1 h at 4 °C with E. coli tRNA (40 µg/ml), heparin (5 mg/ml), DTT (100 mM), and poly(U) RNA
(5 µg/ml) with constant rocking. Extracts were centrifuged at
10,000 × g for 30 min. The extracts were treated with
RNasin (40 units; Promega) and incubated four times for 1 h each
with 100 µl of poly(C)-agarose (Sigma). The poly(C)-agarose with the
bound proteins was washed five times with a 10× volume of wash buffer
(100 mM KCl, 20 mM HEPES pH 7.9, 1 mM DTT, 0.5 mM PMSF, 1 mg/ml heparin) and
eluted with salt gradient (0.5, 1, 1.5, and 2 M KCl, 20 mM HEPES, pH 7.9, 1 mM DTT, and 0.5 mM PMSF) for 15 min each at room temperature. The eluted
fractions were concentrated, washed twice with the wash buffer
(Microcon-10 microconcentrators; Amicon), and resuspended in the
storage buffer (50 mM KCl, 20 mM HEPES, pH 7.9, 10% glycerol, 1 mM DTT, 0.5 mM PMSF).
Electroelution of Proteins from Ribonucleoprotein
Complexes--
The binding reactions were performed using 15 µg of
unlabeled HIPBS oligoribonucleotide and 1.5-2 mg of protein extract
from PC12 cells in a 300-µl reaction volume. The control reactions were identical except for the RNA. Each 300-µl reaction was loaded into a 35-mm2-wide well of a preparative nondenaturing gel.
One well contained the binding reaction with the labeled RNA for
further identification of the RNA·protein complex. The gel was
exposed to x-ray film, and the fragments of gels containing unlabeled
complexes co-migrating at the same level as the labeled complexes were
excised, macerated, and loaded into the elution cell in the inside cup
buffer (5 mM Tris acetate, pH 8, 0.1% SDS, 0.1 mM EDTA) using a protein electroelution unit (CBS
Scientific). Proteins were eluted overnight at 4 °C with a constant
voltage of 100 mV in the outside cup buffer (50 mM Tris
acetate, pH 8, 0.1% SDS, 0.1 mM EDTA). The inside and outside compartments were separated by dialysis membrane with a
molecular weight cut-off of 12,000-14,000 (Spectra/por*2; Spectrum Medical Industries). The eluted proteins were concentrated using Microcon-10 microconcentrators (Amicon), resuspended in the sample buffer, and subjected to SDS-PAGE analysis.
Northwestern and Western Analysis--
Proteins were separated
on 9% SDS-PAGE and transferred onto nitrocellulose with a semidry
blotter (Bio-Rad). The blotted proteins were renatured in buffer
containing 10 mM Tris, pH, 7.5, 50 mM NaCl, 1 mM EDTA, 1× Denhardt's solution, 1 mM DTT,
and 1 mg/ml heparin for 1 h at room temperature. The blots were
then hybridized with 1 × 106 cpm of 5'-labeled HIPBS
oligoribonucleotide in the same binding buffer without DTT or heparin,
but with E. coli tRNA (20 µg/ml), overnight at room
temperature. The blots were then washed briefly in the same buffer and
exposed to x-ray film. For Western blot analysis, blots were stripped
in PBS with 1% SDS for 1 h at 37 °C. The blots were blocked in
Tris-buffered saline + Tween 20 with 5% nonfat milk and were probed
with the primary antibody in Tris-buffered saline + Tween 20 with 5%
dry milk overnight. The signal was visualized by exposing the blots to
chemiluminescence reagents (Amersham). Chicken anti-PCBP antibody that
recognizes PCBP1 and PCBP2 isoforms was a gift
from S. A. Liebhaber (11). A specific rabbit anti-PCBP1
antibody against the peptide sequence of PCBP1 (amino acids
201-220, GenBankTM accession number U24223 (9)) was raised
and affinity-purified by Alpha Diagnostic. A specific
anti-PCBP2 antibody (GenBankTM accession number
X78136) was a gift from A. V. Gamarnik and R. Andino (12).
Statistical Analysis of the Data--
The mRNA half-lives
were calculated for each experiment from linear regression lines fitted
to all time points for each individual experiment. The average
t1/2 is given as mean ± S.D.
(n Stability of TH mRNA Is Decreased by Mutation within the
Pyrimidine-rich Site--
To determine the role of HIPBS in the
regulation of TH mRNA stability, we stably expressed, in PC12
cells, the full-length exogenous TH mRNA with either wild-type or
mutated HIPBS. The exogenous mRNAs were marked by a short sequence
located in the 5'-end of the message
(HindIII-BamHI, Fig.
1A) to differentiate exogenous
from endogenous TH mRNAs and to measure both in the same sample
using RPA (Fig. 1B).
First we analyzed the constitutive expression of exogenous as compared
with endogenous TH mRNA. The steady-state level of exogenous
wild-type TH mRNA was at 64.8 ± 6.9% (n = 5)
of the endogenous TH mRNA (Fig. 1, B, lanes 4 and 5, and C). In contrast, the steady-state
level of the mutated TH mRNA was significantly decreased and was
only 37.3 ± 1% (n = 4, p < 0.01) of the endogenous TH mRNA (Fig. 1, B, lanes
6 and 7, and C).
Next we analyzed degradation rates of the wild-type and mutated
exogenous TH mRNAs after inhibition of transcription with actinomycin D (Fig. 2). During normoxia
the t1/2 of the wild-type TH mRNA was 9.2 ± 0.4 h (n = 3; Fig. 2A, solid squares), and it increased almost 2-fold to 17.2 ± 0.5 h (n = 2, p < 0.01; Fig.
2A, open squares) during hypoxia. This is similar to the half-life measured for endogenous TH mRNA (7.5 ± 0.4 h (n = 5) in normoxia and 15 ± 0.7 h (n = 5) in hypoxia; p < 0.01). The
half-life of the mutated TH mRNA was significantly lower at 4 ± 0.8 h (n = 3, p < 0.01; Fig.
2B, solid squares), and it not only failed to
increase during hypoxia but showed a small decrease to 2.9 ± 0.3 h (n = 5, p = 0.035, Fig.
2B). These data indicate that HIPBS is an mRNA
stabilizing element.
HIPBS Is an mRNA Stabilizing Element in the Context of a
Heterologous mRNA--
To further characterize the role of HIPBS
in regulation of mRNA stability, we stably expressed in PC12 cells
chimeric mRNA that have fragment of TH 3'-UTR containing HIPBS
cloned downstream from the CAT reporter gene (Fig.
3A). The chimeric mRNA had
either a wild-type (CAT-wtTH mRNA) or mutated, inactive
(CAT-mutTH mRNA) HIPBS site.
Steady-state levels of the CAT-wtTH mRNA were
substantially increased compared with CAT mRNA lacking the TH
insert (Fig. 3B, lanes 4-6 compared with
lane 3). In contrast, steady-state levels of the
CAT-mutTH mRNA (lane 7) were significantly
lower than either CAT-wtTH mRNA (lanes 4-6)
or CAT mRNA alone (lane 3). Importantly, the change in
the constitutive levels of chimeric mRNAs was also reflected in the
comparable differences in the levels of the derived protein measured
using CAT assays (not shown).
Measurements of the degradation rates of CAT, CAT-wtTH, and
CAT-mutTH mRNAs after transcriptional inhibition with
actinomycin D (Fig. 4) show that
insertion of the wtTH sequence into the 3'-UTR of the CAT
gene increased the half-life of the chimeric mRNA from 5 ± 0.2 (n = 3) to 11.6 ± 1 h (n = 5, p < 0.01). On the other hand, insertion of the
same sequence with a mutation that abolished protein binding (7)
resulted in a substantial destabilization of the chimeric mRNA and
a decrease in the mRNA t1/2 to 2.3 ± 0.05 h (n = 2). This is significantly lower
(p < 0.01) than the half-life of the
CAT-wtTH mRNA.
To determine whether expression of chimeric CAT-wtTH
mRNA is regulated during hypoxia, the mRNA levels were measured
during normoxia and hypoxia in the absence or presence of actinomycin D. Unexpectedly, hypoxia failed to regulate expression of the CAT-wtTH mRNA in PC12 cells (data not shown). Further,
even insertion of the full-length 3'-UTR and 5'-UTR of TH mRNA
downstream and upstream, respectively, from the CAT gene did not confer
hypoxic regulation to this chimeric mRNA (data not shown). Thus,
the HIPBS is an mRNA stabilizing element that is necessary but not
sufficient for the O2-dependent regulation of
TH mRNA turnover. Moreover, additional regulatory elements involved
in the hypoxic regulation are most likely located within the coding
region of TH mRNA.
Characterization of the RNA·Protein Complex--
The TH
mRNA·protein complexes were analyzed by UV light cross-linking of
TH mRNA·protein-binding reactions and separation by SDS-PAGE
under reducing conditions (100 mM DTT). Two large complexes
were identified at approximately 80 and 50 kDa (Fig. 5A, lane 1). Addition of
poly(U) competitor blocked, to a large extent, the 80-kDa complex,
revealing a major complex at 50-55 kDa (Fig. 5A, lane
2), but it did not affect formation of the complex identified in
the RNA gel shift assays as shown previously (6). Formation of the
50-55-kDa complex in the presence of poly(U) was abolished by addition
of the unlabeled 162-base-long TH transcript, HIPBS
oligoribonucleotide, or poly(C) RNA as competitors (Fig. 5A,
lanes 3-5). A 50-55-kDa complex was formed in the absence of poly(U) when labeled HIPBS oligoribonucleotide was used as a probe
(Fig. 5A, lane 6). The complex was not formed
when the TH mRNA mutated within the protein-binding site was used
in the binding reaction (Fig. 5B). This mutant did not form
a complex detectable in the gel retardation assays (7). The 50-55-kDa complex was induced by hypoxia in PC12 cells 2.6 ± 0.3-fold
(n = 3, p < 0.02) (Fig.
5C). Thus, the 50-55-kDa ribonucleoprotein complex is the
main complex associated with HIPBS and is regulated by hypoxia.
Identification of the Protein Factor in the Complex with the
Pyrimidine-rich Sequence in TH mRNA--
Because poly(C) RNA is
such an effective competitor of the complex formation (6, 7),
poly(C)-agarose affinity was used to purify the binding factor from the
S-100 protein extracts from PC12 cells (Fig.
6A). Proteins bound to
poly(C)-agarose were fractionated by elution with increasing
concentrations of KCl (0.5-2 M). The presence of the TH
mRNA-binding activity was determined by Northwestern assay with
32P-labeled HIPBS (Fig. 6A, left). The 40-kDa
HIPBS-binding protein was identified in the eluates (Fig. 6A,
lanes 2-5). This protein was further identified as the 40-kDa
PCBP (also known as
Protein fractions eluted with the salt gradient were pooled and
analyzed for their ability to form complexes with TH mRNA. The
combined eluted fractions formed a 50-55-kDa complex with TH
transcript that migrated with the same mobility (Fig. 6B,
lane 4) as the complex formed by the total S-100 fraction
(Fig. 6B, lane 2) analyzed by UV cross-linking
and SDS-PAGE. Extracts treated with poly(C)-agarose were depleted of
the TH mRNA-binding activity (Fig. 6B, lane
3).
To confirm that the purified poly(C)-binding protein is present in the
HIPBS-associated protein complex, proteins were electroeluted from the
unlabeled complexes identified by the gel shift assay, separated on
SDS-PAGE, and screened by Northwestern analysis with HIPBS probe,
followed by Western analysis with a specific antibody against the two
isoforms of PCBP (Fig. 7A). As
controls, the proteins were eluted from corresponding gel slices in
which the binding reaction did not include RNA. HIPBS bound to a
protein factor electroeluted from the complexes formed in the presence
of RNA (Fig. 7A, lane 2) but not to the proteins
electroeluted from the gel slices in the absence of RNA (Fig.
7A, lane 1). Subsequent reprobing of the same
blot with an antibody specific for the isoforms PCBP1 and
PCBP2 revealed that both isoforms are actually present in
the complex (Fig. 7A, lanes 4 and 6,
respectively). PCBP2 migrates slightly higher than
PCBP1 detected by Western blot, which may represent some
post-translational modifications and a somewhat higher molecular weight
of PCBP2 (a nine-amino acid difference).
To determine whether hypoxia induces expression of the PCBP isoforms in
PC12 cells, cytoplasmic protein extracts were analyzed by Western blot
using antibodies specific for each isoform. A 2.4 ± 0.3-fold
induction in the expression of PCBP1 was measured in four
independent sets of PC12 protein extracts (Fig. 7B). In contrast, no induction (or inhibition) of PCBP2 was
measured (data not shown). To confirm the increased amount of
PCBP1 protein in the complex, proteins were electroeluted
from the HIPBS-associated complexes formed by normoxic or hypoxic
extracts (Fig. 7C). Indeed, proteins electroeluted from the
complexes formed with the hypoxic extracts showed enrichment for the
PCBP1 (Fig. 7C, lane 2). These data
indicate that the PCBP1 isoform is critical for hypoxic
induction of the 50-55-kDa complex associated with TH mRNA.
The major finding from this study is that HIPBS is an mRNA
stabilizing element required for constitutive and hypoxia-regulated control of TH mRNA. A four-point mutation that abolished the
protein-binding site within the full-length TH mRNA resulted in a
2-fold destabilization of the mutated mRNA and a corresponding
2-fold decrease in mRNA steady-state levels. In addition, mutation
of HIPBS abolished the O2-dependent regulation
of TH mRNA stability. We further demonstrated that a short fragment
of the TH 3'-UTR containing the HIPBS was sufficient to confer
augmented mRNA stability on the heterologous mRNA, which in
turn resulted in augmented steady-state levels of the chimeric mRNA
and derived protein. Importantly, the mutated HIPBS conferred
destabilization to chimeric CAT-TH mRNA. The decrease in the
half-life because of the mutation in the protein-binding site was very
similar for both the chimeric CAT-TH mRNA or full-length exogenous
TH mRNA.
Interestingly, whereas HIPBS is a necessary element for the
O2-dependent stabilization of TH mRNA in
the context of the full-length TH mRNA and whereas it binds a
protein factor in a hypoxia-inducible manner, HIPBS alone is not
sufficient to confer hypoxic regulation to the CAT mRNA. Neither
are the full-length 3'- and 5'-UTRs of TH mRNA. This result is
similar to previously published data regarding hypoxic regulation of
VEGF mRNA stability (13). In that study, the full-length 3'-UTR of
VEGF mRNA did not confer hypoxic regulation on the heterologous
mRNA (13). Thus, it is possible that additional necessary
regulatory elements are located within the coding region of
hypoxia-regulated mRNAs. Work is in progress to identify these additional elements in TH mRNA.
Sequences similar to that in TH mRNA were reported to regulate the
stability of other mRNAs. First, the stability of the
erythroid-specific The increased expression of chimeric CAT-TH mRNA was accompanied by
a corresponding increase in expression of functional protein (data not
shown), an indication that translation of the chimeric mRNA was
most likely not affected. This observation is important because
pyrimidine-rich sequences binding similar protein factors were reported
to regulate translation (12, 16, 17).
Analysis of the RNA·protein complexes after UV light cross-linking
and SDS-PAGE analysis revealed the presence of one major broad
50-55-kDa complex. This finding extends and complements the results of
our previous study (6), in which we detected two TH mRNA·protein
complexes: the major 74- and a minor 53-kDa complex. We currently view
the 50-55-kDa complex as the major one. The importance of this complex
became clear only after competition with poly(U), which blocked protein
binding at 80 kDa, and after analysis of complexes formed specifically
with HIPBS oligoribonucleotide in comparison with the complexes formed
with the longer TH transcript. Thus, the 80-kDa complex most likely
represents nonspecific binding of protein factors to sequences other
than HIPBS fragments within the 162-base-long TH transcript. The
50-55-kDa complex should correspond, after subtracting the molecular
mass corresponding to the 28 bases of RNA (8 kDa), to the protein
factor with an approximate molecular mass of 42-47 kDa.
In view of the strong affinity of the binding proteins for poly(C) RNA,
poly(C) RNA-agarose was used to purify the protein. A similar procedure
was used previously to purify the protein factor binding to the
cytidine-rich elements in Interestingly, we measured an approximately 2-fold induction in
expression of PCBP1 but not PCBP2 in
cytoplasmic extracts obtained from hypoxic cells. That corresponds to
the similar enrichment of PCBP1 in the electroeluates from
the ribonucleoprotein complexes formed with the hypoxic protein
extracts and to the quantitative increase in the complex formation.
This difference results from an absolute enrichment for
PCBP1, as similar increases were measured using total
cellular lysates, and immunocytochemistry did not show any
intracellular translocation in the PCBP1
content.2 This difference in the hypoxic inducibility of
the two isoforms supports the observation that although
PCBP1 and PCBP2 have high degrees of sequence
homology, they show some specific functional differences, such as
efficiency of interacting with AUF1 protein (19) or efficiency to
regulate poliovirus translation (12).
The molecular mechanism by which binding of PCBP to the HIPBS element
regulates mRNA stability, especially in the context of increased TH
mRNA stability during hypoxia, is presently unknown. A possible
mechanism would be protection of a nuclease cleavage site within the
region associated with HIPBS in the TH mRNA. Protein binding to the
wild-type sequence may protect this site from nuclease activity. When
protein binding is prevented by mutation, the site becomes accessible
to cleavage, resulting in a rapid degradation of mRNA. The fact
that a fragment of TH mRNA containing mutated HIPBS confers
decreased mRNA stability to the chimeric mRNA may favor such a
possibility. On the other hand, formation of the complex associated
with the globin mRNA stability element indicates its potential role
in stabilization via the poly(A) tails (20, 21). To evaluate this
possibility, a determination of the length of poly(A) tails of TH
mRNA during normoxia and hypoxia is under way.
INTRODUCTION
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Abstract
Introduction
References
CP or hnRNPE (9-12)),
and we demonstrate that expression of the PCBP1 isoform is
induced by hypoxia in PC12 cells.
EXPERIMENTAL PROCEDURES
-32P]UTP (specific
activity > 800 Ci/mmol; NEN Life Science Products) at 42 °C
for 1 h, heated to 95 °C, and digested with RNase-free DNase. A
single RNA band of appropriate size was obtained by gel purification.
The 18 S probe was labeled to lower specific activity (2.5-3 × 105 cpm/µg) by including 1 mM unlabeled UTP
in the transcription reaction (Maxiscript, Ambion). Ten micrograms of
total RNA were hybridized with 0.2 × 106 cpm of CAT
probe or 0.5 × 106 cpm of TH probe and
0.1 × 106 cpm of 18 S probe in the hybridization
buffer (80% formamide, 40 mM PIPES, pH 6.4, 0.4 M NaCl, 1 mM EDTA) at 42 °C in a dry oven
overnight. Samples were then digested with RNase A/T1 mixture at
37 °C for 30 min, RNases were inactivated with proteinase K, and RNA
was phenol-chloroform-extracted and precipitated. Heat-denatured samples were run on a denaturing polyacrylamide gel (8% acrylamide with 7 M urea) and exposed to x-ray film for 18-48 h.
Radioactive bands were identified and quantified using a PhosphorImager
system (Molecular Dynamics). Constitutive expression of the exogenous TH mRNA is presented as percent of the endogenous TH mRNA
expression measured in the same experiment. Expression of the
endogenous TH and CAT mRNAs was directly normalized to the
radioactivity measured in the 18 S ribosomal band. The measurements of
mRNA half-life (t1/2) were performed using actinomycin D (5 µg/ml) as described previously (5).
-32P]UTP-labeled RNA transcripts (50,000 cpm) or
[
-32P]ATP-labeled HIPBS oligoribonucleotide (20,000 cpm) were incubated on ice for 20 min in 10 mM HEPES, pH
7.9, 3 mM MgCl2, 50 mM KCl, 200 ng/µl Escherichia coli tRNA, 10% glycerol, 1 mM DTT, 0.5 mM PMSF and then treated with RNase
T1 and heparin as described (6-8). For the competition experiments,
the competitor RNA was added at the same time. RNA·protein complexes
were resolved on 7% native acrylamide gel. UV light cross-linking was
performed by irradiating the binding reactions with 1 × 106 J/cm2 UV light (Fisher). Samples were
boiled in SDS-sample buffer with 100 mM DTT and
electrophoresed on 8% SDS-polyacrylamide gels as described previously
(6).
3) or S.E. (n > 3) for all
experiments. Unpaired t tests or analysis of variance were
used to calculate statistical significance between the groups. In a
graphical representation, the logarithmic values of the RNA
concentration for each time point are shown, and a straight line is
fitted for all the points in the experimental group by linear
regression using Origin 3.5 linear regression algorithms.
RESULTS
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Fig. 1.
Steady-state levels of wild-type
(wt) or mutated (mut) exogenous
(ex) TH mRNAs in PC12 cells. A,
restriction map of rat TH cDNA (open bar) in the
pcDNA3 expression vector. Locations of the HIPBS element
(hatched region) with wt and mut
sequences and of the marker sequence used to generate the riboprobe
(bracket) are shown. CMV, cytomegalovirus
promoter; BGH, bovine growth-hormone polyadenylation signal.
B, RPA analysis of expression of wt (lanes
4 and 5) or mut (lanes 6 and 7)
exogenous TH mRNAs (exTH mRNA) as compared with the
endogenous TH mRNA (endTH mRNA). M
(lane 1), RNA markers; FP (lane 2),
free probes for TH and 18 S (arrows); V
(lane 3), only endogenous TH mRNA is detected in PC12
cells transfected with an empty pcDNA3 vector. The bottom
panel showing lighter, exposed 18 S bands indicates equal
loading of total mRNA. C, quantification (mean ± S.E.) of the constitutive expression of wt
(black) or mut (gray) exogenous TH
mRNA analyzed by RPA represented as percent expression of the
endogenous TH mRNA.
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Fig. 2.
Degradation rates of wild-type
(wt) (A) or mutated (mut)
(B) exogenous (ex) TH mRNAs during normoxia
( ) and hypoxia (
). The respective RNA levels were estimated
by RPA at the indicated time points after inhibition of transcription
with actinomycin D and expressed as logarithmic values of ratios of the
mRNA measured at time 0. All experimental points were plotted, and
for each group a straight line was fitted by regression analysis. The
average values (mean ± S.E.) of t1/2 for each
group are given below each graph. The gray boxes
represent the overlap of normoxic and hypoxic points.
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Fig. 3.
Steady-state levels of chimeric wild-type
(wt) or mutated (mut) CAT-TH mRNAs in PC12
cells. A, restriction map of chimeric CAT-TH
cDNA in the pcDNA3 expression vector showing the location of
the HIPBS element (hatched region) and the sequence used to
generate a riboprobe (bracket). B, RPA analysis
of expression of the CAT-wtTH (lanes 4-6) or
CAT-mutTH (lane 7) chimeric mRNAs as compared
with CAT mRNA (lane 3) in independent pools of stably
transfected PC12 cells. FP (lane 1), free probe;
V (lane 2), PC12 cells transfected with an empty
pcDNA3 vector. Bottom, the lighter exposure
of RPA for 18 S RNA is shown to demonstrate similarity in the amount of
mRNA loaded in each lane.
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Fig. 4.
Degradation rates of CAT,
CAT-wtTH, and CAT-mutTH mRNAs in pools of
stably transfected PC12 cells during normoxia. A, RPA
analysis of RNA levels for CAT alone (lanes 1-5) or CAT
containing either wild-type TH sequence
(CAT-wtTH, lanes 6-10) or mutated TH
sequence (CAT-mutTH, lanes 11-15) at
indicated times after inhibition of transcription with actinomycin D
(AD). Lower panels show less exposed 18 S bands
and indicate equal loading of RNA. B, quantification of RNA
concentrations (expressed as logarithmic values of the ratio of RNA at
the indicated test time to the time 0) plotted versus time
after inhibition of transcription with actinomycin D. All experimental
points were plotted for each group ( , CAT;
, CAT-wtTH;
, CAT-mutTH), and a straight line was fitted by
regression analysis. The mean ± S.E. of t1/2
for each group is given below.
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Fig. 5.
Identification of the ribonucleoprotein
complex associated with HIPBS. Binding reactions of TH RNA and
protein factors from the S-100 fraction were UV light cross-linked and
analyzed by SDS-PAGE. A, competition of 80- and 50-55-kDa
TH·protein complexes (lane 1) with 100 ng of each
competitor: poly(U) RNA (lanes 2-5), cold, 162-base-long TH
transcript (TH tr, lane 3), HIPBS
oligoribonucleotide (lane 4), or poly(C) RNA
(lane 5). Only the 50-55-kDa complex was formed when HIPBS
oligoribonucleotide was used as a probe (lane 6).
B, the 50-55-kDa complex (arrow) is formed by
wild-type (WT) TH transcript (lane 1) but not by
TH mRNA mutated in the protein-binding region (MUT, lane
2). C, the formation of the 50-55-kDa complex is
induced in cells exposed to 5% O2 (hypoxia, lane
2) as compared with cells exposed to 21% O2
(normoxia, lane 1).
CP or hnRNPE (10-12)) with a specific
polyclonal antibody (Fig. 6A, lanes 7-10).
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Fig. 6.
Identification of HIPBS-binding proteins
using poly(C) RNA-agarose affinity chromatography. A,
analysis of protein factors eluted from poly(C)-agarose with 0.5-2
M KCl. Proteins were subjected to SDS-PAGE, transferred
onto nitrocellulose membrane, and analyzed by Northwestern
(NW) assay using labeled HIPBS oligoribonucleotide
(lanes 2-5) and by Western with chicken anti-PCBP antibody
(lanes 7-10). Lanes 1 and 6, Northwestern and Western analysis of protein factors in S-100 fraction
(50 µg) before incubation with poly(C)-agarose. B, UV
light cross-linking and SDS-PAGE analysis of the complex formed between
the protein factor eluted from poly(C) with the labeled TH transcript.
FP (lane 1), free probe; lane 2,
pre-poly(C) protein extracts before incubation with poly(C)-agarose;
lane 3, poly(C)-depleted protein extracts after incubation
with poly(C)-agarose; lane 4, poly(C) eluate-pooled protein
fractions eluted from the poly(C)-agarose. Arrow indicates
the 50-55-kDa complex. Bracket indicates mobility of the
free probe.
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Fig. 7.
PCBP1 is induced by hypoxia.
A, analysis of the protein factors electroeluted from an
RNA·protein complex (lanes 2, 4, and 6) or a
control gel slice that contains only proteins but no RNA (lanes
1, 3, and 5) by Northwestern (NW) assay
using labeled HIPBS RNA (lanes 1 and 2) and by
Western (W) using specific anti-PCBP1
(lanes 3 and 4) or anti-PCBP2
(lanes 5 and 6) antibodies, respectively.
B, Western blot analysis of two independent sets of normoxic
and hypoxic cytoplasmic extracts demonstrating the strongest (3-fold,
lanes 1 and 2) and the weakest (1.8-fold,
lanes 3 and 4) induction of the PCBP1
by hypoxia. Lane 5, assessment of the antibody specificity
using antigenic peptide in the Western reaction. C, Western
blot analysis of protein factors electroeluted from the
HIPBS-associated ribonucleoprotein complex formed with proteins from
hypoxic (5% O2, lane 2) as compared with
normoxic (21% O2, lane 1) PC12 cells.
DISCUSSION
2-globin mRNA is regulated by
three short cytidine-rich repeats similar to the TH protein-binding
motif (14). Second, a pyrimidine/cytidine-rich sequence within the
collagen
1(I) mRNA is involved in the regulation of the
stability of this mRNA and forms a similar ribonucleoprotein complex with protein factors (15). Mutations in these elements destabilize globin and collagen
1(I) mRNAs. In our study,
however, we show not only that the mutations destabilize TH mRNA
but also that the short fragment of mRNA containing the
protein-binding site is capable of conferring increased mRNA
stability to a heterologous mRNA.
2-globin mRNA (9, 10). As
expected, we purified the 40-kDa poly(C)-binding protein that
corresponds to PCBP (12), also known as
CP (9, 10) or hnRNPE (17,
18), as identified by the specific antibodies. The eluate from the
poly(C)-agarose restored fully formation of the complex with HIPBS RNA
or the TH transcript in the binding reactions. This finding differs
from previously reported data showing that the poly(C)-eluted proteins
did not restore formation of the complex associated with
2-globin stability element and was thus considered
necessary but not sufficient for the complex formation (10). Further,
formation of the globin-associated complex seems to require additional
protein factors such as AUF1 (hnRNPD) protein (19). So far we did not
find any evidence for the presence of AUF protein in the
HIPBS-associated complex.2
The protein-binding motifs within the TH and
2-globin
mRNAs show some potentially important differences. Whereas the
essential PCBP-binding motifs are very similar in the two mRNAs
(11), in the case of TH mRNA the binding motif is located in the
middle of a long pyrimidine-rich sequence, and in the case of
2-globin mRNA the short binding motifs are separated
by purine-rich sequences. This difference may affect the constitution
of the ribonucleoprotein complex associated with the protein-binding
motifs and result in RNA-specific regulation of mRNA stability.
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ACKNOWLEDGEMENTS |
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We thank S. A. Liebhaber for the anti-PCBP antibody and for comments on the manuscript, A. V. Gamarnik and R. Andino for anti-PCBP2 antibody, R. Kole for reading the manuscript, J. E. Beresh and J. B. Streit for technical assistance, and G. Doerman for preparation of the figures.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants HL51078 and HL58687 and American Heart Association Grant-in-aid 9750110N.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U24223 and X78136.
W. R. P. was supported by National Institutes of Health Training
Grant T32 HL07571 and Grant HL MPDS.
§ To whom correspondence should be addressed: Dept. of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, P. O. Box 670576, Cincinnati, OH 45267-0576. Tel: 513-558-1957; Fax: 513-558-5738; E-mail: Maria.Czyzykkrzeska{at}uc.edu.
The abbreviations used are: TH, tyrosine hydroxylase; UTR, untranslated region; HIPBS, hypoxia-inducible protein-binding site; PCBP, poly(C)-binding protein; RPA, RNase protection assay; CAT, chloramphenicol acetyltransferase; PIPES, 1,4-piperazinediethanesulfonic acid; t1/2, half-life; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis.
2 M. F. Czyzyk-Krzeska, unpublished results.
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REFERENCES |
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