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INTRODUCTION |
Plasminogen activators
(PAs)1 are serine proteases that
catalyze the conversion of the zymogen plasminogen to plasmin, a broad spectrum endopeptidase that is responsible for intravascular
fibrinolysis (1). This protein is also known to play a major role in
biological processes involving localized proteolysis of extracellular
matrix, such as tissue remodeling and tumor cell invasion and
metastasis (2). Type-1 plasminogen activator-inhibitor (PAI-1), a
50-kDa glycoprotein, is a major regulator of plasminogen activation (3, 4). PAI-1 is synthesized in a variety of cell types and its expression
is regulated by growth factors and hormones, including agents that
elevate intracellular cAMP levels (5-8).
In HTC rat hepatoma cells, the cyclic nucleotide analogue 8-bromo-cAMP,
together with the phosphodiesterase inhibitor isobutylmethylxanthine (designated cA), increases tissue type PA activity more than 50-fold primarily by decreasing PAI-1 mRNA by 90% and protein by 60-70%. The decrease in PAI-1 mRNA is due to a 60% decrease in the rate of
PAI-1 gene transcription and, more importantly, a 3-fold increase in
the rate of PAI-1 mRNA decay (8, 9). Utilizing HTC cells stably
transfected with chimeric constructs containing portions of the mouse
-globin gene and rat PAI-1 cDNA, the 1730-nucleotide (nt) PAI-1
3'-untranslated region (UTR) (nt 1331-3060) was shown to contain
sequences that mediate the cA-induced destabilization (10).
Furthermore, results obtained from deletion and insertion analyses
using a series of
-globin coding region:PAI-1 3'-UTR chimeric
constructs demonstrated that at least two regions within the PAI-1
3'-UTR mediate the cA effect. One of these regions is the 3'-most 134 nt, from position 2926 to 3060, of the PAI-1 mRNA. This 134-nt
sequence includes a 75-nt U-rich region present at its 5' end and a
24-nt A-rich region at its 3' end (Fig. 1A) (10, 11).
The decay rates of many mRNAs have been shown to be regulated by a
variety of external stimuli including hormones, growth factors, and
agents that elevate intracellular cAMP levels (see Refs. 12 and 13 for
review). Studies aimed at elucidating the mechanism(s) involved in
regulating mRNA stability have identified a number of potential
cis-acting sequences and/or trans-acting factors
(12, 13); however, the molecular basis for the cyclic nucleotides
regulation of mRNA stability remains largely undefined.
The aim of the present study was to further elucidate the mechanism(s)
involved in the cyclic nucleotide-induced destabilization of PAI-1
mRNA in rat HTC cells. To this end, studies were conducted to
identify the cytosolic factors that bind to the 3'-most 134 nt of the
PAI-1 mRNA, to characterize the specificity and binding sites for
these factors, and to determine their role in cA-induced PAI-1 mRNA
destabilization. Results from ultraviolet (UV) cross-linking analyses
demonstrate that specific RNA-protein complexes of ~38, 50, 53, 61, 65, and 76 kDa form with the 134-nt sequence, while RNA electrophoretic
mobility shift analyses (R-EMSAs) demonstrate the formation of high
molecular mass multiprotein complexes. The 50-, 61-, and 76-kDa and
multiprotein complexes form between the A-rich region and HTC cell
cytosolic proteins that are found in both polysomal and post-ribosomal
fractions, while the 38-kDa complex forms between the U-rich region and
proteins found in the polysomal fraction. Mutations in the A-rich
region abolished formation of the 50, 61, and 76 kDa and multiprotein
complexes as well as the ability of cA to regulate the decay of
transcripts from stably transfected globin:PAI-1 chimeric contructs,
suggesting that these RNA-protein complexes play an important role in
the cA-induced destabilization of PAI-1 mRNA.
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EXPERIMENTAL PROCEDURES |
Materials--
8-Bromo-cAMP, isobutylmethylxanthine,
benzamidine,
trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane
(E64), heparin (sodium salt), RNase T1, and tRNA (from Bakers' yeast,
type X) were purchased from Sigma. Leupeptin,
4-(2-aminoethyl)-benzoylsulfonyl fluoride-hydrochloride (Pefabloc®SC (AEBSF)), proteinase K, RNase inhibitor, and
T3 RNA polymerase were obtained from Boehringer-Mannheim. Coomassie®
Plus Protein Assay reagent was acquired from Pierce (Rockford, IL).
Eagle's minimal essential medium, RNase A, RNase T2, T4 DNA ligase, T4 DNA polymerase, restriction enzymes, Benchmark® prestained
molecular weight markers, okadaic acid, and calyculin A were purchased
from Life Technologies (Gaithersburg, MD). AmpliscribeTM T3
transcription kit was obtained from Epicentre Technologies (Madison,
WI). Polyadenylic (poly(A)), polycytidylic (poly(C)), polyguanadylic
(poly(G)), and polyuridylic (poly(U)) acids were acquired from
Pharmacia Biotech. Centricon® concentrators were purchased from Amicon
(Beverly, MA). [
-32P]UTP (800 Ci/mmol) and
[
-32P]ATP (400 Ci/mmol) were obtained from Amersham.
Prestained SDS-polyacrylamide gel electrophoresis (PAGE) standards were
from Bio-Rad.
Cytoplasmic Extract Preparation--
Monolayer cultures of rat
HTC hepatoma cells were grown and maintained as described previously
(9). Cells were incubated in serum-free medium for 10 to 16 h,
harvested by trypsinization, pelleted, and resuspended in 25 mM Tris (pH 7.9), 0.1 mM EDTA, 1 mM
Pefabloc®SC (AEBSF), 1 µM leupeptin, 1 mM benzamidine, 1 µM E64. The cell pellets
were subjected to three freeze/thaw cycles (10 min/cycle) followed by
centrifugation at 4 °C at 10,000 × g for 15 min.
The supernatant fraction (S10) was removed, assayed for protein
concentration using Coomassie® Plus Protein Assay reagent
as directed by the manufacturer, and stored at
70 °C. Isolation of
polysomal and post-ribosomal (S130) extracts was performed essentially
as described (14) with additional proteinase inhibitors added to buffer
A (1 mM Pefabloc®SC (AEBSF), 1 µM leupeptin, 1 mM benzamidine, 1 µM E64). The S130 fraction was concentrated using
Centricon-3® concentrators as instructed by the
manufacturer. The polysomal and S130 fractions were assayed for protein
concentration and stored at
70 °C.
Preparation and Radiolabeling of RNA Probes--
The DNA
template for transcribing the PAI-1 RNA probes 2926-3060 (the
full-length 134-nt region of the 3'-UTR) and 2926-2966 was prepared as
follows: pKS
3'-PAI, which contains the PAI-1 3'-UTR
subcloned into the XbaI site of the pBluescript
KS
(pKS
) multicloning site (MCS), was
digested with KpnI (pKS
: MCS) and
ApaI (PAI-1: bp 2926), incubated with T4 DNA polymerase to
generate blunt ends, and religated using T4 ligase. PAI-1 2926-3060 or
2926-2966 template DNA was linearized with XbaI or
NdeI, respectively, prior to in vitro
transcription. The DNA template for generating the PAI-1 probe
2966-3060 was prepared by digesting pKS
3'-PAI with
KpnI and NdeI (PAI-1: bp 2966), creating blunt
ends using T4 DNA polymerase, and religating using T4 ligase. The
template was linearized with XbaI prior to in
vitro transcription. The DNA template for PAI-1 sequence
2125-2296 was generated by deletion of the SalI
(pKS
: MCS)-NcoI (PAI-1: bp 2125) fragment from
pKS
3'-PAI, blunt end formation and ligation, and
linearization with DdeI (PAI-1: bp 2296) prior to in
vitro transcription. pKS
CAT, which contains
chloramphenicol acetyltransferase (CAT) coding region sequence (bp
2528-2829) subcloned into the EcoRI and SmaI sites of the pKS
MCS was digested with BamHI
(pKS
: MCS) prior to in vitro transcription.
The DNA templates used to generate PAI-1 RNA probes 2926-3024,
3010-3060, and 3024-3060 were generated by polymerase chain reaction
(PCR). Sequence for the T3 RNA polymerase promoter
(AATTAACCCTCACTAAAGGG) was included at the 5' end of the forward primer
and used with the appropriate reverse primer in PCR reactions with pSVL
G/P (globin coding region/PAI-1 3'-UTR; Ref. 10) as template DNA. The
DNA templates for 2926-3060 containing the A-rich region mutations were also generated by PCR (see Fig. 5A for description of
mutations) by incorporating the mutant sequences into the reverse PCR
primer. The DNA templates used to generate the wild-type and mutant
3010-3040 (Fig. 5A) were prepared by annealing
complementary oligonucleotides containing the sequence for the T3 RNA
polymerase promoter at the 5' end followed by the appropriate PAI-1 sequence.
Preparation of radiolabeled RNA probes was performed essentially as
described (15) by incubating the DNA template DNA (oligo- or
PCR-generated templates: 100-200 ng, linearized plasmid templates: 0.5-1.0 µg) with 10 units of T3 RNA polymerase under the following conditions: 40 µM Tris-HCl (pH 8.0, 20 °C), 6 mM MgCl2, 2 mM spermidine, 20 mM dithiothreitol, 0.4 mM CTP, 0.4 mM GTP, 37.5 µM ATP, 37.5 µM
UTP, 10 units of RNase inhibitor, 100 µCi of
[
-32P]UTP (800 Ci/mmol), 50 µCi of
[
-32P]ATP (400 Ci/mmol). To generate unlabeled
competitor RNA, in vitro transcription from the template
DNAs was carried out using the AmpliscribeTM T3 transcription kit as
directed by the manufacturer.
R-EMSA and UV Cross-linking Analyses--
For R-EMSA, extracts
and 32P-radiolabeled RNA were incubated for 20 min at
25 °C in buffer containing 8 units of RNase inhibitor, 10 µg of
yeast tRNA, 10 mM Hepes (pH 7.6), 5 mM
MgCl2, 40 mM KCl, 5% glycerol, and 1 mM dithiothreitol. RNase T1 (1 unit/µl) was added to each reaction and incubation continued at 25 °C for 10 min.
Heparin (5 mg/ml) was added and the reactions were incubated at
25 °C for an additional 10 min. The reactions were subjected to
electrophoresis through a 5% nondenaturing polyacrylamide gel (80:1
acrylamide:bisacrylamide, 40 mA, 4 °C, 4 h) and visualized by
autoradiography. For UV cross-linking analyses, extracts and 32P-radiolabeled RNA were incubated and treated with RNase
T1 and heparin as described for R-EMSA. The reactions were
then exposed to a UV light source (UV Stratalinker 1800, Stratagene) at
a distance of 2.5 cm from the light source for 10 min (1.8 µJ/cm2) unless otherwise specified. RNase A (10 mg/ml)
and RNase T2 (100 units/ml) were added and incubation continued at
25 °C for 10 min; a combination of RNases was utilized in order to
maximize digestion of the unbound radiolabeled RNA probe. SDS sample
buffer was added and reactions were heated at 85 °C for 2 min;
RNA-protein complexes were analyzed by 0.1% SDS-10% PAGE (38:1
acrylamide:bisacrylamide, 40 mA, 25 °C, 4 h) followed by
autoradiography. For competition analyses, S10 proteins were
preincubated with unlabeled competitor RNA for 10 min at 25 °C prior
to the addition of radiolabeled RNA. Each R-EMSA or UV cross-linking
analysis figure presented is representative of at least three
independent experiments.
Analysis of Chimeric Globin:PAI-1 mRNA Stability in HTC
Cells--
Cell culture and maintenance, stable transfections,
construction of pSVL G/G+2925/3054, riboprobe preparation, and
ribonuclease protection analyses were conducted as previously reported
(10). To prepare the chimeric construct pSVL G/G+2925/3054 double
mutant, the PAI-1 sequence from nt 2925-3054 was amplified by PCR,
digested with BglII, and ligated into the BglII
site of pSVL G/G (10). The forward primer contained BglII
sequences at its 5' end, while the reverse primer contained the two
mutations in the A-rich region (nt 3023-3028; AAAAAAA changed to
cccccc and nt 3030-3035 AUAAA changed to ccgccc) and BglII
sequences at its 5' end.
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RESULTS |
Interaction of HTC Cell Cytosolic Proteins with the cA-responsive
3'-most 134 nt of the PAI-1 3'-UTR--
To identify potential
mRNA-binding proteins in HTC cell cytosolic extracts that interact
with the 3'-most 134 nt of the PAI-1 3'-UTR (nt 2926-3060), UV
cross-linking analyses were conducted. Using radiolabeled nt 2926-3060
and HTC cytoplasmic extracts, a major ribonucleoprotein complex with an
Mr of 50,000 (doublet) and minor complexes of
38,000, 53,000, 61,000, 65,000, 76,000, and 86,000 were detected (Fig.
1B, lane 1). Formation of the
RNA-protein complexes was dependent on protein concentration and on the
time of exposure to UV light; no additional interactions were detected with greater than 30 µg of extract or after 5 min of UV exposure (data not shown). To ensure complete formation of RNA-protein interactions, all subsequent reactions were exposed to UV light for 10 min (1.8 µJ/cm2).

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Fig. 1.
UV cross-linking and R-EMSA analyses of HTC
cell cytoplasmic extracts and radiolabeled PAI-1 3'-UTR sequence
2926-3060. A, the nucleotide sequence of the PAI-1 mRNA
from position 2926-3060 is represented. Highlighted are the U-rich
(underline) and A-rich (bold underline) regions
located between nt 2948-3022 and nt 3023-3046, respectively.
B, HTC cell S10 extract (30 µg) was incubated with
radiolabeled nt 2926-3060 (1 × 105 cpm; 0.4 ng) and
UV cross-linking analysis was carried out as described under
"Experimental Procedures" with the following modifications:
lane 1, no modification; lane 2, proteinase K
(0.5 mg/ml) was added to the reactions during the heparin incubation;
lane 3, radiolabeled RNA was incubated at 85 °C for 15 min followed by rapid cooling on ice; lane 4, S10 extract
was incubated at 85 °C for 15 min followed by rapid cooling on ice.
The Mr (in thousands) of the RNA-protein
complexes as based on molecular weight standards is indicated.
C, HTC cell S10 extract (30 µg) was incubated with
radiolabeled nt 2926-3060 (2 × 104 cpm; 0.08 ng) and
analyzed by R-EMSA as described under "Experimental Procedures"
with the following modifications: lane 1, no modification;
lane 2, proteinase K (0.5 mg/ml) was added to the reactions
during the heparin incubation; lane 3, radiolabeled RNA was
incubated at 85 °C for 15 min followed by rapid cooling on ice;
lane 4, S10 extract was incubated at 85 °C for 15 min
followed by rapid cooling on ice. The approximate size of the
RNA-protein complexes indicated (Mr in
thousands) is based on the migration of protein standards.
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Ribonucleoprotein complex formation was abolished when the samples were
treated with proteinase K or when S10 proteins were denatured by
heating the extract prior to incubation with radiolabeled RNA (Fig.
1B, lanes 2 and 4, respectively). No complexes
were detected when the RNA alone was subjected to UV cross-linking analysis. These results confirm that the complexes are comprised of RNA
and protein. Heating the RNA for 15 min at 85 °C prior to incubation
with protein had no effect on complex formation (Fig. 1B, lane
3). Identical ribonucleoprotein complexes were seen when longer
in vitro generated transcripts (nt 2714-3060 or nt
2502-3060) that included the 3'-most 134 nt were used as probes in UV
cross-linking (data not shown).
R-EMSAs using the 3'-most 134 nt of the PAI-1 3'-UTR (nt 2926-3060)
demonstrated the formation of an abundant high molecular mass
RNA-protein complex of approximately 175 kDa (determined by comparison
with protein molecular mass markers) and a minor complex of
approximately 140 kDa (Fig. 1C, lane 1). Complex formation was protein concentration-dependent and was abolished when
proteinase K was added to the reaction or when S10 proteins were
denatured prior to incubation with radiolabeled probe (Fig. 1C,
lanes 2 and 4, respectively). No complexes were
detected in the absence of extract (data not shown). These results
indicate that the complexes are comprised of RNA and protein. Heating
the RNA prior to incubation with protein had no effect on complex
formation (Fig. 1C, lane 3).
Subcellular Distribution of Cytoplasmic RNA-binding
Proteins--
To determine the subcellular distribution of the
cytoplasmic proteins that interact with PAI-1 sequence 2926-3060, UV
cross-linking analyses were performed using S10, polysomal, or S130
fractions as the source of proteins. The 38-kDa complex was formed with polysome-associated proteins, but not with S130 proteins; the remainder
of the RNA-protein complexes formed using proteins found in both the
polysomal and S130 fractions. Using equal amounts of protein from the
polysomal and S130 fractions, the majority of the mRNA binding
activity was in the polysomal fraction. Likewise, the multiprotein
complexes detected by R-EMSA formed with proteins found in both the
polysomal and S130 fractions (data not shown).
Specificity of RNA-Protein Complex Formation--
The specificity
of the interactions detected between HTC cell cytoplasmic proteins and
the radiolabeled PAI-1 sequence 2926-3060 was demonstrated by
competition UV cross-linking analyses and R-EMSA (Fig.
2). Extract was preincubated with unlabeled
competitor RNA prior to the addition of radiolabeled PAI-1 probe.
Unlabeled PAI-1 sequence 2926-3060 as competitor reduced formation of
the RNA-protein complexes detected by both UV cross-linking (Fig. 2A, lanes 1-4) and R-EMSA (Fig. 2B, lanes 1-5)
in a concentration-dependent manner, indicating that these
interactions are specific. A portion of the PAI-1 3'-UTR (nt
2125-2296), which when inserted into the 3'-UTR of the
-globin gene
fails to confer cA-responsiveness (10), was not able to compete with
labeled 2926-3060 for complex formation (Fig. 2, A, lane 6;
B, lane 7). Likewise, there was no competition for most of
the complexes when CAT RNA was used as a nonspecific competitor (Fig.
2, A, lane 5; B, lane 6); however, the 86-kDa
complex may be nonspecific as CAT RNA did compete for its binding.
Furthermore, no RNA-protein interactions were detected when HTC cell
S10 extract was incubated with radiolabeled PAI-1 sequence 2125-2296
or CAT RNA (data not shown), supporting the conclusion that the
observed interactions are specific for the 134-nt cA-responsive
sequence.

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Fig. 2.
Specificity of RNA-protein complex formation
between HTC cytoplasmic proteins and radiolabeled PAI-1 3'-UTR sequence
2926-3060. A, unlabeled PAI-1 nt 2926-3060 (lanes
1-4, 0, 10, 50, 100-fold molar excess) or 250-fold molar excess
unlabeled CAT (lane 5) or PAI-1 nt 2125-2296 (lane
6) was preincubated with 30 µg of S10 proteins prior to
incubation with radiolabeled nt 2926-3060 (1 × 105
cpm). RNA-protein complexes were analyzed by UV cross-linking analysis
as described under "Experimental Procedures." B,
unlabeled PAI-I nt 2926-3060 (lanes 1-5, 0, 10, 50, 100, 250-fold molar excess) or 250-fold molar excess unlabeled CAT
(lane 6) or PAI-I nt 2125-2296 (lane 7) was
preincubated with 30 µg of S10 proteins prior to incubation with
radiolabeled nt 2926-3060 (2 × 104 cpm). RNA-protein
complexes were analyzed by R-EMSA as described under "Experimental
Procedures." The Mr (in thousands) of the
RNA-protein complexes is indicated.
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Since PAI-1 sequence 2926-3060 contains a 75-nt U-rich region and a
24-nt A-rich region (Fig. 1A), competition UV cross-linking analyses and R-EMSAs were performed using unlabeled homoribopolymers as
competitor RNAs. In the UV cross-linking studies (Fig.
3A), formation of the 38-kDa
complex was inhibited by the presence of poly(U), but not poly(A). In
contrast, formation of the 50-, 61-, and 76-kDa complexes was inhibited
by the presence of poly(A), but not poly(U); the 38-, 53-, and 65-kDa
complexes remained after competition with poly(A). Poly(C) and poly(G)
did not inhibit formation of any of the observed complexes. These data
suggest that the 38-kDa complex forms with PAI-1 sequences contained
within the U-rich region and that the 50-, 61-, and 76-kDa complexes form with the A-rich sequence between nt 3023 and 3046. In the R-EMSA
studies (Fig. 3B), formation of the RNA-protein complexes was abolished by the presence of poly(A), but was unaffected by poly(U), poly(G), or poly(C). These results suggest that the
multiprotein complexes and the major complexes observed by UV
cross-linking require the same A-rich sequence.

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Fig. 3.
Effect of competitor homoribopolymers on
RNA-protein complex formation between HTC cytoplasmic proteins and
radiolabeled PAI-1 3'-UTR sequence 2926-3060. A, unlabeled
competitor RNA at 0, 10, or 100-fold molar excess was preincubated with
30 µg S10 protein prior to incubation with radiolabeled nt 2926-3060
(1 × 105 cpm). RNA-protein complexes were analyzed by
UV cross-linking analysis as described under "Experimental
Procedures." B, unlabeled competitor RNA at 0, 10, or
100-fold molar excess was preincubated with 30 µg of S10 proteins
prior to incubation with radiolabeled nt 2926-3060 (2 × 104 cpm). RNA-protein complexes were analyzed by R-EMSA as
described under "Experimental Procedures." The
Mr (in thousands) of the RNA-protein complexes
is indicated.
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Identification of the Sequences Involved in RNA-Protein Complex
Formation--
To further define the nucleotide sequences to which the
cytoplasmic proteins bind, radiolabeled RNA probes corresponding to those diagrammed in Fig. 4A were
generated and used in UV cross-linking analyses. In each case, molar
equivalents of each radiolabeled RNA were used (Fig. 4B).
S10 proteins and nt 2966-3060 formed complexes with the same migration
as those observed using nt 2926-3060. Cytoplasmic proteins and nt
3010-3060 or 3024-3060, containing the A-rich region, also formed
complexes essentially the same as those observed using 2926-3060,
except that the 38-kDa complex was absent. When S10 proteins were
incubated with 3010-3040, the abundant complexes migrating at 50 kDa
were present as well as faint complexes at 53, 61, 65, and 76 kDa; the
38-kDa complex was absent.

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Fig. 4.
Identification of the sequences involved in
RNA-protein complex formation. A, schematic of the
radiolabeled RNA probes. B, HTC cell S10 extract (30 µg)
was incubated with radiolabeled probe and analyzed by UV cross-linking
analysis as described under "Experimental Procedures." Reactions
contained molar equivalents of each RNA probe: 1 × 105 cpm of 2926-3060 (lane 1), 7.6 × 104 cpm of 2926-2966 (lane 2), 3.8 × 104 cpm of 3010-3060 (lane 3), 5.5 × 104 cpm of 3024-3060 (lane 4); 2.8 × 104 cpm of 3010-3040 (lane 5), 7.6 × 104 cpm of 2926-3024 (lane 6), or 2.4 × 104 cpm of 2926-2966 (lane 7). C,
HTC cell S10 extract (30 µg) was incubated with radiolabeled probe
and analyzed by R-EMSA as described under "Experimental
Procedures." Reactions contained 2 × 104 cpm of
2926-3060 (lane 1), 1.5 × 104 cpm of
2926-3024 (lane 2), or 5.6 × 103 cpm of
3010-3040 (lane 3).
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Incubation of cytoplasmic proteins with nt 2926-3024 (Fig. 4B,
lane 6), which contains only the U-rich region, resulted in formation primarily of the 38-kDa complex; only faint bands appeared at
~50, 53, and 65 kDa. This pattern of RNA-protein interactions was
also observed using another 3'-UTR U-rich sequence located upstream at
nt 2790-2911 as probe (data not shown). S10 incubated with nt
2926-2966 resulted in no RNA-protein complex formation (Fig. 4B,
lane 7).
As a correlate to these studies, radiolabeled 2926-3024 or 3010-3040
were used as probes in R-EMSAs to determine if the A-rich region was
also important for formation of the multiprotein complexes detected by
native PAGE. As shown in Fig. 4C, the complexes detected using nt 2926-3060 also form with nt 3010-3040, but not with nt 2926-3024 (compare lanes 2 and 3 with lane
1). These data indicate that the multiprotein complexes form using
the same A-rich region as those ribonucleoprotein complexes detected by
UV cross-linking analyses.
Together with results from homoribopolymer competition assays, these
studies suggest that (i) the majority of the RNA-protein complexes
detected by both R-EMSA and UV cross-linking analysis are the result of
an interaction of cytoplasmic proteins with a sequence containing the
A-rich region (nt 3023-3046) and (ii) only formation of the 38-kDa
RNA-protein complex detected by UV cross-linking analysis results from
specific interactions between S10 proteins and a sequence containing
the U-rich region (nt 2948-3022).
Mutational Analysis of the A-rich Region--
Since the U-rich
region of the 134-nt sequence alone does not confer cA-responsiveness
onto the otherwise non-responsive globin mRNA (10) and the majority
of the RNA-protein interactions occur within the A-rich region,
mutational analyses were performed on the A-rich region. Mutations were
generated in the context of the 134-nt PAI-1 sequence (nt 2926-3060)
or a 30-nt sequence containing the A-rich region (nt 3010-3040), as
illustrated in Fig. 5A. Mutation of the A-rich region (mutant I: nt 3023-3028, AAAAAA to cccccc and
mutant II: nt 3030-3035, AAUAAA to ccgccc) results in loss of RNA
binding activity when the mutations were present in either RNA context
(Fig. 5B), suggesting that the A-rich region located between
nt 3023-3035 is necessary for formation of the 50-, 61-, 65-, and
76-kDa complexes. In addition, formation of the multiprotein complexes
detected by R-EMSA also requires this region as indicated by the lack
of complex formation using radiolabeled 2926-3060 containing either
mutation (Fig. 5C). Mutations in the A-rich region made in
the context of the 134-nt sequence also decreased formation of the
38-kDa complex (Fig. 5B, lanes 2 and 3).

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Fig. 5.
Effect of mutations in the A-rich region on
RNA-protein complex formation. A, schematic of the wild-type
and mutant A-rich region radiolabeled RNA probes. B, HTC
cell S10 extract (30 µg) was incubated with radiolabeled probe and
analyzed by UV cross-linking analysis as described under
"Experimental Procedures." Reactions contained 1 × 105 cpm of wild-type, mut I, or mut II 2926-3060
(lanes 1-3) or 2.8 × 104 cpm of
wild-type, mut I, or mut II 3010-3040 (lanes 4-6).
C, HTC cell S10 extract (30 µg) was incubated with
radiolabeled probe and analyzed by R-EMSA as described under
"Experimental Procedures." Reactions contained 2 × 104 cpm of wild-type, mut I, or mut II 2926-3060
(lanes 1-3). I, nt 3023-3028 (AAAAAA to
cccccc); II, nt 3030-3035 (AAUAAA to ccgccc).
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Role of the A-rich Region in cA-regulated PAI-1 mRNA
Stability--
HTC cells were stably transfected with a chimeric
construct containing the wild-type cA-responsive PAI-1 fragment
inserted into the 3'-UTR of the murine
-globin gene (pSVL
G/G+2925/3054 (10)) or pSVL G/G+2925/3054 containing both A-rich region
mutations (pSVL G/G+2925-3054 double mutant). HTC cells were incubated
in the absence or presence of cA and the decay rates of the chimeric mRNAs were determined as described previously (10). The top panel of Fig. 6 shows the gel from an
RNase protection assay of one such experiment and the bottom
panel shows graphically the pooled data from two experiments. The
wild-type 130-nt fragment mediated cA-induced destabilization of the
globin:PAI-1 chimeric mRNA; in contrast, the mutant 130-nt fragment
failed to confer cA-responsiveness onto the
-globin gene. Consistent
with previous reports (10), control constructs pSVL G/G (globin coding
region/globin 3'-UTR) and pSVL G/P (globin coding region/PAI-1 3'-UTR)
showed no cA responsiveness and a 2-fold increase in mRNA turnover
in response to cA, respectively (data not shown). These results
strongly suggest that the same A-rich region that interacts with HTC
cell cytoplasmic proteins mediates cyclic nucleotide-induced
destabilization of mRNA in HTC cells.

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Fig. 6.
Effect of mutations in the A-rich region on
cA-regulated mRNA stability in HTC cells. HTC cells were
stably transfected with the chimeric constructs pSVL GG+2925/3054
(WT) or pSVL GG+2925/3054 double mutant (Mutant),
which contains both mutants I and II as described in the legend to Fig.
5, and analyzed for cyclic nucleotide regulation of mRNA stability
as described previously (10). The transfected cells were incubated in
the presence or absence of 1 mM 8-Br-cAMP plus 1 mM isobutylmethylxanthine and RNA harvested at the times
indicated. RNA from the transfected genes was assayed by RNase
protection analysis (40 µg of RNA per sample). The top
panel shows the RNase protection data from a single experiment;
duplicate lanes represent duplicate cultures. The sizes of
the protected fragments are: globin, 119 nt; glyceraldehyde phosphate
dehydrogenase (GAP), 80 nt. Size markers in lanes
M are DNA from HpaII-digested and
32P-labeled pBR322. RNase protection assays were quantified
by PhosphorImager analysis. The value for globin in each sample was
normalized to the signal obtained for GAP and the data are expressed as
percent of RNA at time 0. The data shown in the bottom panel
represent the average (± S.E.) of two independent experiments, each
containing duplicate cultures at each time point. , control; ,
cA.
|
|
 |
DISCUSSION |
In HTC rat hepatoma cells, cA causes a 3-fold increase in the rate
of degradation of PAI-1 mRNA; the 3'-most 134 nt of the PAI-1
mRNA is sufficient to mediate the major part of this effect (10).
To better understand the mechanism by which cA induces destablization
of PAI-1 mRNA, studies were conducted to identify trans-acting factors that interact with the 134-nt
cA-responsive sequence, to characterize the specificity and binding
sites of these interactions, and to determine their role in the
regulation of mRNA stability. UV cross-linking analysis
demonstrated HTC cytoplasmic mRNA-binding proteins of approximately
38, 50, 53, 61, 65, and 76 kDa, and R-EMSA demonstrated multiprotein
complexes of ~175 and 140 kDa that interact with the cA-responsive
134-nt sequence. The 38-kDa mRNA-binding protein appears to
interact with the U-rich region. Its binding is competed by poly(U),
and the 38-kDa complex forms with PAI-1 mRNA containing U-rich
sequences between nt 2966 and 3024, but not with the A-rich region (nt
3024-3060). However, complex formation is markedly decreased by
mutations in the A-rich region, suggesting that conformational changes
or protein-protein interactions with other mRNA-binding proteins can influence formation of the 38-kDa complex. The binding site for the
majority of the mRNA-binding proteins (50, 53, 61, 65, and 76 kDa)
and the multiprotein complexes was limited to a 30-nt sequence
containing the A-rich region. Finally, and most importantly, through
mutational analyses the A-rich region was determined to be necessary
for both RNA-protein interaction and for regulation of mRNA
stability in HTC cells. Thus, these studies link the binding of
cytoplasmic proteins to an A-rich region in the PAI-1 3'-UTR with the
cyclic nucleotide regulation of PAI-1 mRNA stability in HTC cells.
Neither the mobility nor the abundance of the complexes formed between
the 134-nt sequence and HTC cytosolic proteins, however, appears to be
regulated by cA. The mRNA binding activity of cytosolic proteins
isolated from HTC cells incubated with cA for 30, 60, 90, 120, 180, or
240 min was essentially the same as that of controls. In addition, the
distribution of complexes between the polysome and S130 fractions did
not appear to be altered after incubation with cA (data not shown).
Several hypotheses may explain the observed lack of cA-regulated
ribonucleoprotein complex formation. First, there may be subtle changes
in complex formation that are not detectable within the limits of the
assays performed in this study. Second, the protein components of the
complexes may be modified in response to cA; for example,
protein-protein interactions within the multiprotein complexes might be
altered by cA without affecting their migration through a nondenaturing
gel. Third, additional proteins that are induced or repressed by cA,
but do not alter RNA-protein complex formation, may be necessary for
the regulation of mRNA stability. Detection of these co-factors by
R-EMSA may be difficult if their interaction with the multiprotein
complexes is weak. Finally, the presence of cA may alter the function
of a protein(s) by allosteric or active site modifications without affecting its ability to form ribonucleoprotein complexes. This modification would be reminiscent of the regulation of cAMP-responsive element (CRE)-binding protein (CREB) by protein kinase A (16). Phosphorylation of CREB at Ser-133 by protein kinase A enhances the
transactivation potential of CREB; however, it has no effect on the
binding of CREB to a consensus CRE suggesting separate regulation of
CREB binding and transcriptional activity.
The stability of a variety of mRNAs is subject to regulation by
intracellular cAMP levels. For example, lactate dehydrogenase A subunit
(18), tyrosine aminotransferase (19), renin (20),
2-adrenergic receptor (21), osteocalcin (22), the
glucose transporter, GLUT1 (23), chorionic gonadotropin (24),
phosphoenolpyruvate carboxykinase (25), and the RII
subunit of
protein kinase A (26) mRNAs are stabilized in the presence of cAMP,
cAMP analogues, and/or cAMP elevating agents. Conversely, the messages
for PAI-1 (9), adrenodoxin reductase (27), and tyrosine hydroxylase (28) are destabilized in the presence of cAMP, cAMP analogues, and/or
cAMP elevating agents. A number of receptor mRNAs are also destabilized by elevated intracellular cAMP levels (29-37). Despite the importance of cAMP as a regulator of mRNA stability, the
mechanism by which increases in intracellular cAMP levels induce
changes in mRNA turnover rates remains undefined in most systems
described to date.
Limited studies, however, have identified potential cis- and
trans-acting mediators of cAMP-regulated mRNA stability.
In hamster smooth muscle cells, the cAMP elevating agent isoproterenol
or the cAMP analogue CPT-cAMP destabilizes
2-adrenergic
receptor (
2-AR) mRNA and induces the binding of the
Mr 35,000
2-AR binding protein
(
-ARB), to a 20-nt region in the 3'-UTR (34). The 20-nt AU-rich
sequence, which contains an AUUUUA motif flanked by U-rich regions, was
shown to mediate the agonist and cAMP-induced destabilization of
2-AR mRNA. A nonconsensus AU-rich nonamer
(UAAUAUAUU) in the human
-AR 3'-UTR that binds the hamster
-ARB
in vitro was shown to be a critical determinant for the
isoproterenol-induced destabilization of
2-AR
transcripts in transfected human embryonic kidney cells (35).
In contrast, a cytosine-rich region in the coding region of the
luteinizing hormone/human chorionic gonadotropin receptor mRNA,
which is destabilized during hCG-induced down-regulation, was found to
form an Mr 50,000 ribonucleoprotein complex with rat ovary cyotsolic proteins; complex formation was enhanced in the
down-regulated state (37). Finally, phosphoenolpyruvate carboxykinase
mRNA is stabilized by dibutyryl-cAMP, CPT-cAMP, or 8-bromo-cAMP in
FTO-2B rat hepatoma cells (25, 38). CPT-cAMP also decreases the binding
of a 100-kDa cytosolic protein to a region in the 3'-UTR that
contains alternating purine:pyrimidine bases, numerous repeats and
palindromes; binding was shown to be sequence-independent, requiring
RNA secondary structure for complex formation.
The cis-acting sequences described in this report are unlike
those described for other systems in which mRNA stability is regulated by intracellular cAMP levels. First, the sequence that mediates the cA-induced destabilization of PAI-1 mRNA involves a
predominantly A-rich region located at the extreme 3' end of the PAI-1
3'-UTR. This is in contrast to the C-rich region implicated in the cA
regulation of leutinizing hormone/human chorionic gonadotropin receptor
mRNA (37) and the primarily U-rich region containing AUUUA or
nonconsensus AU-rich motifs that has been implicated in the regulation
of
2-AR mRNA stability (34-36) and a number of
other regulated mRNAs (39-45). Second, RNA-protein complex
formation between the cA-responsive PAI-1 sequence (nt 2926-3060) or a
truncated sequence containing the A-rich region (nt 3010-3040,
3010-3060, or 3024-3060) is not abolished by heating the RNA at
85 °C for 15 min, followed by rapid cooling (Fig. 1, B
and C; and data not shown). This is in contrast to the
cis-acting sequences involved in cAMP-regulated
phosphoenolpyruvate carboxykinase mRNA stability (38); in this
system, heating the RNA probe prior to UV cross-linking significantly
reduced binding. However, because RNA can rapidly regain secondary
structure after being heated, these experiments cannot rule out a role
for RNA secondary structure in the binding we observe.
The identity of the mRNA-binding proteins described in this report
is unknown. Because the target sequence of most of these proteins is
A-rich, the possibility that poly(A)-binding protein is involved must
be considered. Poly(A)-binding protein, which is highly conserved
across species, has a molecular mass of about 70,000 (46); in contrast,
the major protein binding to the PAI-1 3'-UTR has a mass of about
50,000.
Degradation of mature mRNAs is a regulated process that can have a
significant, and rapid, impact on gene expression. The half-lives of
mRNAs in eukaryotic cells can range from minutes for highly
regulated gene products such as proto-oncogenes, growth factors, and
cytokines to many hours for very stable species such as globin (see
Refs. 12 and 13 for review). Regulation of mRNA stability, often
but not necessarily in conjunction with changes in transcription rates,
allows the level of a particular mRNA to be increased rapidly
and/or transiently in response to various stimuli (13). In most cases,
however, the mechanism(s) by which these stimuli exert their effects
are not clear; it is not known whether these stimuli act directly or
indirectly to cause an increase or decrease in mRNA decay rates.
The studies presented here provide further insight into the mechanism
by which cyclic nucleotides regulate PAI-1 mRNA stability in rat
HTC hepatoma cells. The minimal sequence that interacts with HTC
cytoplasmic proteins was limited to a 30-nt sequence that contains an
A-rich region. This same A-rich region, by mutational analysis, was
shown to be critical for cA-regulated PAI-1 mRNA destabilization in HTC cells. Studies are currently directed at (i) isolating,
identifying, and characterizing the mRNA-binding proteins and the
protein components of the multiprotein complexes and (ii) determining
how the cytoplasmic proteins interact with the A-rich region to elicit
the cA-induced destabilization of PAI-1 mRNA. The ability to link
RNA-protein complex formation with the regulation of mRNA stability
by cyclic nucleotides in HTC cells provides a valuable sytstem in which to study cis- and trans-acting mediators of
regulated mRNA stability.