From the Department of Medicine, Monash University, Box Hill Hospital, Box Hill 3128, Victoria Australia and the § Laboratory for Cancer Medicine, Centre for Medical Research, School of Medicine and Pharmacology, Royal Perth Hospital Unit, Western Australian Institute for Medical Research, the University of Western Australia, Perth 6000 Western Australia, Australia
Received for publication, December 20, 2002, and in revised form, February 6, 2003
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ABSTRACT |
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Plasminogen activator inhibitor type 2 (PAI-2) is
a serine protease inhibitor that is subject to regulation at the
post-transcriptional level. At least two mRNA instability elements
reside within the PAI-2 transcript; one in the coding region and
another within the 3'-untranslated region (UTR). For the latter, a
functional AU-rich motif (ARE) has been identified that provides a
binding site for a number of cellular proteins, including the mRNA
stability protein, HuR. In this study, we used the yeast three-hybrid
system to screen a human leukocyte cDNA library to identify other
proteins that associate with the PAI-2 ARE. This screen identified
tristetraprolin (TTP) as a PAI-2 mRNA ARE-binding protein. UV
cross-linking and immunoprecipitation experiments showed that TTP
expressed in HEK293 cells could associate with the PAI-2 ARE in
vitro. Co-transfection of plasmids expressing TTP and
PAI-2 in HEK293 cells resulted in an increase in the decay rate of
PAI-2 mRNA and loss of PAI-2 protein in a process that was
dependent upon the PAI-2 3'-UTR. The 29-nt PAI-2 AU-rich element alone
was also capable of conferring TTP-dependent mRNA
instability to a reporter transcript. The extent of PAI-2 mRNA
stability was remarkably sensitive to TTP since TTP-dependent PAI-2 mRNA decay occurred at TTP levels
that were below Western blot detection limits. This study
identifies TTP as a functional PAI-2 ARE-binding protein that modulates
the post-transcriptional regulation of the PAI-2 gene.
Plasminogen activator inhibitor 2 (PAI-2)1 is a member of the
plasminogen activator family of proteins and controls the activity of
urokinase plasminogen activator (u-PA) in the extracellular compartment
(1). PAI-2 is also a member of the OV-serpin family of serine protease
inhibitors and has been considered as the most enigmatic member of this
group of proteins (2). One of the most striking features of PAI-2 is
that it exists in two molecular forms: a predominant non-glycosylated
protein and a less abundant secreted glycosylated protein. The
predominant intracellular location of PAI-2 has suggested additional
functions for this u-PA inhibitor, and growing evidence has indicated
an involvement for PAI-2 in the intracellular events associated with
differentiation (3), proliferation (4, 5), signal transduction (6) and
possibly apoptosis (7, 8). PAI-2 has also generated interest because of
its impressive regulatory profile. PAI-2 gene transcription rates are markedly increased in response to the tumor promoter phorbol
12-myristate 13-acetate (PMA) (9), the phosphatase inhibitor, okadaic
acid (10), tumor necrosis factor- Notwithstanding the important contribution of transcriptional control
(14, 15) to the regulation of PAI-2 expression, significant regulation
also occurs at the post-transcriptional level, most notably at the
level of PAI-2 mRNA stability (16, 17). Post-transcriptional
regulation of gene expression has proven to be a particularly important
component in the global control of gene regulation. It is now well
established that the 3'-untranslated region (3'-UTR) of mRNA is
critical in the decision-making process of transcript longevity (18).
Most studies have focused on conserved AU-rich elements within the
3'-UTR of unstable cytokine or proto-oncogene transcripts. Typical
elements are mostly of the AUUUA or UUAUUUAUU type or variants thereof
(19, 20), and are mRNA destabilizing motifs that act, in part, by
their ability to attract specialized RNA-binding proteins to elicit the
decay process. These same elements have also been linked to other
aspects of mRNA metabolism and processing including RNA trafficking
(21) and translation (22), indicating that a given AU-rich element has
pleiotropic effects.
A number of AU-rich element-binding proteins have been identified and
shown to interact with these elements in many of these unstable
transcripts including AUF-1 (23), AUH (24), the ELAV protein family
members (25), and tristetraprolin (TTP) (26). AUF-1 for example,
engages AU-rich elements in a variety of these genes, and the affinity
of interaction correlates with the ability of these sequences to serve
as mRNA instability elements (27). Members of the ELAV family
including HuD, HuC, and HuR (HuA) can also interact with these same
elements, but at least for HuR, this interaction is associated more
with stabilization rather than destabilization of mRNA (28-30).
Arguably the most powerful mRNA destabilizing protein identified is
TTP. This protein is a critical regulator of TNF- Previous studies have addressed the mechanisms underlying the
post-transcriptional regulation of the PAI-2 gene. Two
functional mRNA instability elements have been identified within
the PAI-2 transcript, one within the 3'-UTR of PAI-2 mRNA (16, 17), and the other within exon 4 of the coding region (34). Closer analysis
of the 3'-UTR region of PAI-2 mRNA led to the identification of a
nonameric AU-rich element (UUAUUUAUU) (ARE), 304-nt upstream from the
poly(A) tail, as a mRNA destabilizing determinant (16). This
element was subsequently shown to provide a binding site for a number
of cytoplasmic and nuclear proteins (17), one of which was HuR,
although the role of HuR in the post-transcriptional control of PAI-2
remains to be determined. To identify and characterize additional
proteins that assemble on the PAI-2 ARE, a human leukocyte cDNA
library was screened for PAI-2 AU-rich element-binding proteins using
the yeast three-hybrid approach. This screen identified tristetraprolin
(TTP) as a specific PAI-2 ARE-binding protein that associates with the
PAI-2 AU-rich element and participates in the PAI-2 mRNA decay
process. Since TTP is widely considered as a post-transcriptional
regulator of cytokine gene expression in response to inflammatory
signals our study suggests that the biological targets of TTP are not
restricted to cytokines and implicates PAI-2 in a broader context of
the inflammatory response associated with TTP.
Yeast Strains and Plasmid Constructs--
The reporter yeast
strain L40-coat and the RNA bait plasmid vectors pIIIA/MS2-1 and
pIIIA/MS2-2 (containing selection marker URA3, ADE2) (35,
36) were generous gifts from Dr. Marv Wickens of the University of
Wisconsin. The TTP mammalian expression plasmid CMV.hTTPtag (31)
(hereafter referred to as pTTP-HA) was provided by Dr. Paul Bohjanen
(University of Minnesota). The wild-type PAI-2 expression plasmid
pCI-PAI-2 contains the 1872-bp PAI-2 cDNA (9) inserted into the
EcoR1 site of the expression plasmid pCl-neo (Promega,
Madison, WI) (34).
The RNA bait plasmids were constructed as follows. The
5'-phosphorylated oligonucleotides of the ARE region (29 bp) of the human PAI-2 gene, were synthesized by Sigma Genosys:
5'-TTTTACTTTGTTATTTATTATTTTATATA-3' and
5'-TATATAAAATAATAAATAACAAAGTAAAA-3'. After annealing, the double-stranded ARE oligonucleotide was inserted into the
SmaI site of the pIIIA/MS2-2 and pIIIA/MS2-1, creating
ARE/MS2-2 and ARE/MS2-1, respectively (36). The insertion and
orientation of the ARE in the plasmids was confirmed by PCR and
sequence analysis (sequencing primer:
5'-CTGTCTCTATACTCCCCTATAG-3').
Construction of Globin/PAI-2 3'-UTR
Constructs--
Plasmid pCMV-glo (hereafter referred to as pCI-glo),
contains the rabbit Screening cDNA Library Using the Yeast Three-hybrid
System--
The yeast three-hybrid screening procedures were performed
essentially as described elsewhere (35, 38, 39). The plasmids pIIIA/ARE-MS2-2 and pIIIA/ARE-MS2-1 were introduced into yeast L40-coat
by transformation. A human leukocyte cell cDNA library (250 µg;
Clontech) was transformed into L40/pIIIA/ARE-MS2-2
and L40/pIIIA/ARE-MS2-1. Double transformants (Trp+,
Leu+, Ura+) were then plated onto Trp
As a negative selection procedure, candidate yeast clones were
transferred onto plates containing 0.1% 5-fluoro-orotic acid (5-FOA)
to eliminate the majority of RNA-independent false positive colonies
(38). The same yeast clones were also transferred onto Trp
In order to test candidate colonies for RNA sequence-specific
activation of HIS3, the RNA dropout colonies expressing the putative RNA-binding protein cDNA, were retransformed with four different RNA plasmids: ARE/MS2 Cell Culture and Gene Transfection--
HEK293 cells were
maintained in Dulbecco's modified Eagle's medium (DMEM) containing
10% heat-inactivated fetal calf serum, 2 mM glutamine, and
1× penicillin and streptomycin at 37 °C under 5% CO2.
HEK293 cells were transiently co-transfected with the PAI-2 expression
plasmid pCI-PAI-2 (17) and the TTP expression plasmid pTTP-HA,
or globin/PAI-2 3'-UTR and TTP expression plasmids, by the calcium
phosphate method (40), except that the transfection mixture was allowed
to incubate with the cells for 16-24 h, and the glycerol shock was
omitted (31). Fresh DMEM medium containing 10% heat-inactivated fetal
calf serum was then added and cells maintained for a further 24 h.
Total RNA was extracted for Northern blotting as described below.
Cross-linking of Protein to RNA and
Immunoprecipitation--
Bluescript plasmids harboring the 29-nt PAI-2
AU-rich element (17) were used to transcribe the PAI-2 ARE RNA
probes in vitro. Following linearization with
XbaI, 1 µg of template was incubated for 2 h at
37 °C in the presence of 50 µCi of [
To prepare protein extracts for the UV cross-linking and
immunoprecipitation, confluent cells were collected by trypsinization, washed three times with phosphate-buffered saline, then lysed for 5 min
on ice in 100 µl/106 cells of cytoplasmic extraction
buffer (CEB; 10 mM HEPES, pH 7.1, 3 mM
MgCl2, 14 mM KCl, 0.2% Nonidet P-40, 5%
glycerol, 1 mM dithiothreitol, 2 µg/ml aprotinin, 0.5 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 1 mM sodium orthovanadate, and 1 mM NaF). The
nuclei were pelleted for 1 min at 1000 × g at 4 °C, and the supernatant containing the cytosolic fraction was aliquoted, snap frozen in liquid nitrogen, and stored at
For the binding assays, 4 µg of protein extract were preincubated
with 150 µg of heparin and 40 units of RNase inhibitor (RNasin; Promega) for 10 min at room temperature in CEB buffer before addition of the RNA probe (200 cps) in a total volume of 20 µl. After a further 30 min incubation at room temperature, samples were transferred to a 96-well microplate, which was placed on ice and irradiated for 15 min at a distance of 5 cm from a UV source (Ultra LUM model UVB-20).
RNA not associated with protein was digested with 100 units of RNase T1
(Roche Molecular Biochemicals) for 20 min at room temperature. In some
experiments the RNase T1-resistant RNA-protein complexes were further
digested with 1 µg of RNase A (Roche Molecular Biochemicals) at
37 °C for 15 min.
The samples were diluted to 0.4 ml in CEB buffer, and protein
A-Sepharose (Amersham Biosciences) added (total volume of 0.45 ml).
These were incubated overnight at 4 °C in the presence of either
preimmune rabbit serum (1:150 dilution) or crude polyclonal rabbit
anti-HA (HA.11) antiserum (1:150 dilution) (BabCO, Richmond, CA).
Immune complexes were recovered by centrifugation, washed three times with CEB buffer, resuspended in 50 µl of 2× SDS sample buffer, and subjected to SDS-PAGE on 10% acrylamide gels and autoradiography.
Northern Blotting--
Total RNA was purified from transfected
cells as described by Chomczynski and Sacchi (42). 20-µg aliquots of
RNA were electrophoresed through 1% agarose gels containing 20%
formaldehyde and subsequently transferred to Hybond-N+ membranes
(Amersham Biosciences). The filters were hybridized with
32P-labeled DNA probes as described (17). Membranes were
processed by standard techniques and exposed to Kodak BioMax film
(Eastman Kodak) at
The labeled cDNA probes used for hybridization were obtained as
follows: 752 bp of the exon 1-6 PCR-amplified fragment of PAI-2
cDNA (9); the 1.1-kb HindIII TTP cDNA fragment of
pTTP-HA (CMV.hTTPtag (31)); and the 380-bp
HindIII/BamHI fragment of pCMV-glo containing the
globin cDNA fragment (37).
Western Blotting--
Western blotting was performed as
previously described (34). Cytoplasmic extracts (up to 50 µg)
prepared from cells were separated by SDS-PAGE under reducing
conditions and transferred to nylon membranes. Membranes were
hybridized with antibodies directed against TTP (1:1000 dilution; kind
gift from Dr. Andrew Clark) or PAI-2 (1:10,000 dilution; American
Diagnostica). Secondary antibodies coupled to horseradish peroxidase
were added (1:2,000 dilution) and the immunocomplexes
assessed by enhanced chemiluminescence.
Yeast Three-hybrid Screening for PAI-2 AU-rich Element-binding
Proteins--
The yeast three-hybrid approach was used to screen a
human leukocyte cDNA library for proteins that interacted with the
functional 29-nt AU-rich element within the 3'-UTR of PAI-2 mRNA.
This genetic approach yielded seven positive clones that interacted
with the PAI-2 AU-rich RNA bait in both an RNA-dependent
and RNA sequence-dependent manner. Sequence analysis
revealed that three of seven clones were identical, and all clones
contained partial cDNA sequences (1399-1436 bp) that were
homologous to human TTP (Fig. 1).
The 5'-end point of the clones ended between TTP Interacts with the PAI-2 AU-rich Element in Vitro--
To
confirm that TTP could directly interact with the PAI-2 ARE, RNA probes
containing a single copy of the PAI-2 AU-rich element were incubated
with cytoplasmic extracts prepared from either mock-transfected HEK293
cells, or cells transiently transfected with 5 µg of plasmid pTTP-HA.
Samples were UV cross-linked then immunoprecipitated with anti-HA
antibodies or preimmune serum, and subjected to SDS-PAGE (Fig.
2, lanes 1-4). Replicate
samples were digested with RNase T1 either alone (lanes
5-8) or in combination with RNase A (lanes 9-12)
prior to immunoprecipitation. As shown, extracts prepared from cells
transfected with 5.0 µg of pTTP-HA did not produce an
immunoprecipitable complex in the presence of preimmune serum
(lane 2). However, protein-RNA complexes were detected when
samples were immunoprecipitated with the anti-HA antibodies (lane
4). Following RNase T1 digestion, two complexes of ~120 and 55 kDa still remained (lane 8). When samples were digested with
both RNase T1 and RNase A only a single complex of molecular mass 52 kDa was present (lane 12), although the 120-kDa complex was
still present after longer exposure of gel to x-ray film. The 120-kDa
complex most likely represents TTP associating with another protein and
is more sensitive to RNase A possibly due to conformational changes.
The slight reduction in molecular mass of the HA-containing RNA complex
from 55 to 52 kDa is consistent with the further trimming of the RNA
probe from the HA-tagged TTP protein by RNase A. Extracts derived from
mock-transfected cells did not contain any HA-containing immune
complexes. Taken together, these data indicate that TTP expressed in
cells recognizes the AU-rich element in the PAI-2 3'-UTR.
Overexpression of TTP Destabilizes PAI-2 mRNA in HEK293
Cells--
It was important to determine whether TTP could influence
the post-transcriptional regulation of the PAI-2 gene. TTP
has previously been shown to influence cytokine gene expression by
associating with AU-rich elements and accelerating the decay rate of
the cytokine transcript. To determine whether TTP could act in a
similar manner on PAI-2 mRNA, plasmids expressing PAI-2 (pCI-PAI-2)
and TTP (pTTP-HA) were co-transfected into HEK293 cells. In this
experiment, cells were transfected with a fixed amount of pCI-PAI-2 (5 µg) and increasing amounts (0, 0.005, 0.01, 0.05, 0.1, 1.0, 5.0 µg)
of plasmid pTTP-HA.
As shown in Fig. 3 panel A,
cells transfected with pCI-PAI-2 expressed a PAI-2 transcript
(lane 2, middle blot). However, the intensity of
this transcript decreased as the expression levels of TTP increased
(lanes 3-9). It is also evident that levels of PAI-2
mRNA were significantly reduced at relatively low levels of TTP
mRNA expression (e.g. lane 6). Furthermore,
higher concentrations of TTP resulted in the formation of a stable but
truncated PAI-2 transcript (lanes 7-9; indicated with
arrow). The significance of the formation of this truncated
PAI-2 transcript is unclear, but it is unlikely to be of physiological
significance since this occurs only at levels of very high TTP levels.
It is interesting to point out that high concentrations of TTP have
also been shown to produce a stable truncated transcripts of TNF,
GM-CSF, and IL-3 (43).
TTP Expression Correlates with Reduced Levels of PAI-2
Protein--
To confirm that the reduced levels of PAI-2 mRNA
following TTP expression correlated with a reduction in the synthesis
of PAI-2 protein, a similar experiment was performed as described in
Fig. 3 (panel A), and total cellular protein extracted and assessed for PAI-2 and TTP protein by SDS-PAGE and Western blotting. As
shown in panel B, increasing levels of TTP protein
correlated with decreasing levels of PAI-2 protein. It is also
important to note that the levels of expressed TTP that conferred a
marked reduction in PAI-2 protein occurred at levels of TTP that were below the detection limit of the Western blot assay (lane 4 of panel B). This suggests that PAI-2 mRNA is
exquisitely sensitive to TTP; only a relative low concentration of TTP
is sufficient to impart significant instability to PAI-2 mRNA and
loss of PAI-2 protein biosynthesis.
The PAI-2 3'-UTR Confers TTP-mediated Instability to a Reporter
Transcript--
To determine whether TTP regulates PAI-2 mRNA
stability via the PAI-2 3'-UTR, we determined the ability of the
full-length PAI-2 3'-UTR to destabilize a globin reporter transcript in
a TTP-dependent manner. HEK293 cells were transfected with
a constant amount (5 µg) of pCI-glo-PAI-2 and increasing amounts of
pTTP-HA (0-5 µg). As shown in Fig. 4,
panel A, the intensity of chimeric globin-PAI-2 mRNA
levels decreased as the level of TTP increased in cells. In this
experiment, TTP was more effective at destabilizing the chimeric
globin-PAI-2 transcript than PAI-2 mRNA itself, since globin-PAI-2
mRNA levels were already substantially reduced in cells transfected
with 0.01 µg of pTTP. This was observed in at least three independent
experiments and suggests that stability elements outside the PAI-2
3'-UTR can modulate the destabilizing effect of TTP on PAI-2 mRNA.
Also, akin to the results presented in Fig. 3, panel A,
higher concentrations of TTP resulted in the accumulation of a
truncated but stable globin-PAI-2 mRNA intermediate (indicated by
arrow to the right of Fig. 3). The effect of TTP on the stability of the chimeric transcript was due to the presence of
PAI-2 3'-UTR sequences, because TTP had no effect on globin transcripts
devoid of the PAI-2 3'-UTR (panel B).
The PAI-2 AU-rich Element Independently Confers
TTP-dependent mRNA Instability--
To determine
whether the PAI-2 AU-rich element could independently confer
TTP-mediated mRNA instability, HEK293 cells were transiently
co-transfected with plasmids expressing the globin transcript alone or
containing either a single or tandem copies of the 29-nt PAI-2 AU-rich
element, together with increasing amounts of pTTP-HA. As seen in Fig.
5, globin transcripts devoid of
additional 3' sequences were not destabilized by increasing levels of
TTP (lanes 1-5). In contrast, globin transcripts containing
the single copy of the PAI-2 AU-rich element were destabilized by TTP
in a concentration-dependent manner (lanes
7-11). It is also apparent that the ability of the AU-rich
element to confer TTP-dependent destabilization was less
efficient than seen with the full-length PAI-2 3'-UTR (compare with
Fig. 4, panel A). Interestingly, globin transcripts
harboring two copies of the PAI-2 AU-rich element conferred
TTP-dependent mRNA destabilization more effectively than the single copy AU-rich element (compare lanes 7-11
with lanes 13-17). These results indicate that the ARE
within PAI-2 3'-UTR partially conveys the mRNA destabilizing
function of TTP. However, since the single copy AU-rich element was not
as effective as the full-length PAI-2 3'-UTR, it appears likely that
complete TTP effect requires engagement with additional elements within the PAI-2 3'-UTR to implement the decay process.
PAI-2 is subject to post-transcriptional regulation, and a
significant component of this regulation relies on a functional AU-rich
element within the 3'-UTR. In this study, we used the yeast
three-hybrid approach to identify tristetraprolin as a PAI-2 AU-rich
element-binding protein. TTP, also known as TIS11, G0S24, Nup475
(44-48), is the product of the immediate-early response genes Zfp-36
in mouse and ZFP36 in humans. TTP has previously been described as an
mRNA destabilizing protein that associates with AU-rich elements
within the 3'-UTR of cytokines, most notably TNF, GM-CSF, and IL-3 (26,
31, 43) and promotes deadenylation subsequent to mRNA degradation.
TTP contains two zinc finger domains that have been shown to be
necessary for TTP to interact with AU-rich elements in cytokine
transcripts. Each of the five distinct TTP cDNA clones that were
found to recognize the PAI-2 AU-rich element by the yeast three-hybrid
approach were incomplete clones, but all contained both zinc finger
domains. This is consistent with the possibility that the two zinc
finger domains within the TTP molecule are also necessary for TTP to
recognize the PAI-2 AU-rich element. One of the TTP clones was found to
contain a 43-bp deletion at the 3'-end of the transcript. Whether this
TTP variant defines a new closely related member of the TTP family of
proteins requires further investigation.
UV cross-linking and immunoprecipitation assays demonstrated that TTP
expressed in HEK293 cells formed an RNase T1- and RNase A-resistant
52-kDa complex with a single copy of the PAI-2 ARE. The size of the
complex was consistent with that of the HA-tagged TTP protein coupled
to the partly digested RNA probe. TTP was active in the turnover of
PAI-2 mRNA decay since co-transfection of HEK293 cells with a PAI-2
expression plasmid with increasing amounts of a TTP expression plasmid
resulted in destabilization of the PAI-2 transcript in a
TTP-dependent manner. Furthermore, the effect of TTP was
concentration-dependent, since the degree of PAI-2
instability correlated with an increase in the expression of TTP. In
fact, PAI-2 mRNA levels were particularly sensitive to TTP since
PAI-2 mRNA levels were substantially reduced in cells transfected
with as little as 0.1 µg of pTTP-HA. Destabilization of PAI-2
mRNA by TTP was also accompanied with a concomitant reduction in
the level of PAI-2 protein as assessed by Western blotting and in these
experiments, PAI-2 protein was essentially abolished when cells were
co-transfected with 0.1 µg of pTTP-HA. The 3'-UTR of PAI-2 was also
shown to be necessary and sufficient to convey TTP-mediated instability
while the PAI-2 AU-rich element in isolation was able to partially
confer TTP-dependent destabilization to the globin reporter
gene. The fact that the AU-rich element alone was less effective than
the full-length PAI-2 3'-UTR at conferring TTP-dependent mRNA destabilization suggests
that TTP requires additional elements within the PAI-2 3'-UTR to
implement the decay process. Nonetheless, this study clearly identifies
TTP as a potent post-transcriptional regulator of PAI-2 gene expression.
Curiously, overexpression of TTP resulted in the accumulation of a
truncated PAI-2 transcript that appeared to be relatively stable. The
significance of this is presently unclear, although overexpression of
TTP as well as other members of this family of mRNA-binding
proteins has been shown to produce stable truncated variants of TNF,
GM-CSF, and IL-3 (43). This truncation has been previously mapped to
occur within the TNF AU-rich element (43). As judged by the size of the
truncated PAI-2 transcript, it also likely that the truncation is
missing sequences 3' of the PAI-2 AU-rich region. This was further
supported from Northern blot data using probes specific for regions
immediately 5' or 3' of the AU-rich element, because only the 5'-probe
hybridized with the truncated transcript (data not shown). We also
suspect that this truncated transcript is unlikely to be of
physiological significance since this effect was only seen in the
presence of very high levels of TTP, levels unlikely to be achieved
in vivo. Furthermore, truncations of these transcripts,
including PAI-2, have not been reported in any physiological
circumstance and as such are unlikely to be generated under natural
conditions. It has been proposed that the formation of these stable
truncated intermediates under high concentrations of TTP is a
consequence of a sequestration of mRNA decay proteins by TTP, or
due to excessive loading of TTP onto the transcript thereby preventing
the exosome-mediated decay process (31).
Mice with a targeted deletion of the TTP gene display a
complex inflammatory syndrome (26) consisting of inflammatory
arthritis, dermatitis, cachexia, autoimmunity, and myeloid hyperplasia.
TTP One of the long standing questions in the control of
post-transcriptional gene expression is how transcripts that contain similar functional AU-rich elements and also provide binding sites for
a common collection of RNA-binding proteins, still display a distinct
pattern of regulation. In some cases, other regulatory domains have
been shown exist within these transcripts that might act to temper the
effect of the powerful AU-rich regulatory domains. On the other hand,
although the same RNA-binding proteins can engage these transcripts, it
is possible that the means by which these proteins modulate specific
transcripts may differ for example, in the requirement for ancillary
cofactors or specific phosphorylation events. The means by which TTP
elicits its destabilizing effect on PAI-2 is unclear although it is
likely that some of the requirements seen in the regulation of TNF by
TTP would be similar for PAI-2. Nonetheless the fact remains that the
time course of PAI-2 mRNA and protein accumulation is vastly
different from that seen for TNF, and hence PAI-2 mRNA must contain
additional regulatory domains (see below) or attracts other proteins to
counteract the destabilizing effect of TTP in vivo.
We have identified TTP as an important regulator of PAI-2
gene expression, thereby extending the involvement of TTP beyond that
of cytokine regulation. This therefore raises the possibility that
PAI-2 may be an integral part of the inflammatory response since PAI-2
appears to be regulated by the same agents that regulate TNF. Our
previous studies on the role of the AU-rich element within the PAI-2
3'-UTR led to the identification of the mRNA stabilizing protein
HuR as a prominent PAI-2 AU-rich element-binding protein (17). Although
there is a lack of direct evidence to implicate HuR in the
stabilization of PAI-2 mRNA, it is tempting to speculate that HuR
and TTP act in an opposing manner to maintain steady state levels of
PAI-2 mRNA. Such a scenario has been postulated for the regulation
of the GM-CSF transcript by HuR and TTP (49). Although clearly
implicated in the regulation of PAI-2 gene expression, these
proteins may not necessarily function in isolation, since a number of
other unidentified proteins also associate with the PAI-2 AU-rich
element and are likely to influence the destabilizing/stabilizing actions of TTP and HuR. This picture is further complicated by the fact
that PAI-2 mRNA turnover is also influenced by instability elements
residing within exon 4 of the PAI-2 coding region. A 28-nt sequence
within exon 4 provides a binding site for an unidentified protein of 52 kDa. The role of this protein in the control of PAI-2 mRNA
stability and its possible association with proteins associating with
the PAI-2 AU-rich element is unknown, but it would not be surprising if
some interaction occurs between PAI-2 mRNA-binding proteins to
these instability elements.
The PAI-2 transcript is inherently unstable but the degree of
instability can be modulated by agents that either increase (e.g. phorbol esters or Dioxin) or decrease (e.g.
glucocorticoids) PAI-2 expression. It would be interesting to determine
the role of TTP during modulation of PAI-2 mRNA stability by these
agents and also to determine the effect of silencing of TTP expression (for example by siRNA) on the rate of PAI-2 mRNA accumulation under
steady-state and regulated conditions.
Our previous REMSA studies using HT-1080 cells have shown that a number
of cellular factors recognize the PAI-2 AU-rich element and during
PMA+TNF treatment a transient increase in the binding activity of one
particular mRNA- binding protein is observed (34). The time course of
this transient increase in binding activity parallels the accumulation
of TTP mRNA in the same cells (data not shown) raising the
possibility that the transiently induced PAI-2 mRNA-binding protein
is TTP itself. However, we have been unable to convincingly demonstrate
that TTP is indeed the protein in question because of the limitation of
the anti-TTP antibodies presently available.
In summary, we have identified TTP as a post-transcriptional regulator
of PAI-2 gene expression that associates with the PAI-2 AU-rich element and implements the decay process. It would be interesting to determine whether disruption of TTP expression in cells
changes the induction profile of the PAI-2 gene, thereby establishing a direct physiological role for TTP in PAI-2
gene expression in vivo. Further studies are required to
determine the mechanisms by which TTP implements PAI-2 mRNA decay,
and if there is any difference in the mechanism by which TTP
orchestrates the decay of its target transcripts.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TNF) (11, 12) and
lipopolysaccharide (LPS) (13).
and GM-CSF
mRNA instability (26, 31, 32). This has been decisively born out in
TTP
/
mice, which develop a severe inflammatory phenotype because
of the increase in cytokine expression resulting from the loss of
negative regulation at the post-transcriptional level (26). TTP is
therefore clearly a mRNA destabilizing protein that acts at least
in part by promoting deadenylation and by attracting the exosome to the
transcript for rapid decay (33).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-globin gene driven by the CMV promoter (37). Plasmid pCI-glo-PAI-2 is the same as pCI-glo, but contains the entire
3'-UTR of the PAI-2 mRNA inserted into the 3'-UTR of the globin
reporter gene (16). Plasmid pCI-glo-ARE and pCI glo-2ARE are the same
as pCI-glo, but contain either a single copy or two copies of the 29-nt
PAI-2 ARE inserted into the 3'-UTR of the globin reporter gene,
respectively. To prepare these plasmids, complementary oligonucleotides
containing the single (29 nt) or tandem (58 nt) copy of the PAI-2
AU-rich element were synthesized. These oligonucleotides also included
restriction sites for XhoI at each end to enable subcloning
into the XhoI site of pCI-glo. Oligonucleotides were
annealed, digested with XhoI, and inserted into the
XhoI site of pCI-glo using standard techniques.
, Leu
,
His
, Ura
plates containing 5 mM 3-aminotriazole
(3-AT).
, Leu
,
His
Ura
plates containing 5 mM 3-AT ("master
plates"). The 5-FOA-sensitive colonies identified on the master
plates were subsequently patched onto non-selective plates (Trp
,
Leu
) to select for colonies in which the RNA plasmid had been removed ("RNA-dropout colonies"): After 3-4 days growth, single yeast colonies that had the RNA plasmid dropped out (pink color) were spotted
back onto Leu
Trp
plates containing 0.1% 5-FOA to confirm that the
RNA plasmid had been removed and were able to grow. In parallel, yeast
were also transferred to Leu
, His
plates containing 5 mM 3-AT to retest for the RNA-dependent
activation of HIS3.
2, ARE/MS2
1, PAI-2 exon 4/MS2
2 as
an unrelated RNA bait, and RNA bait vector pIIIA/MS2-2. After transformation, the single yeast colonies were picked and spotted onto
Ura
, Trp
, Leu
plates to confirm the success of transformation and
also onto Trp
, Ura
, Leu
, His
plates containing 5 mM
3-AT to test for RNA sequence-specific activation of HIS3.
Positive colonies should grow only with RNA bait plasmids containing
the PAI-2 ARE sequence, but not with baits containing irrelevant RNA sequence or empty RNA bait vector alone. cDNA plasmids from
positive clones were isolated and subjected to DNA sequencing analysis (GAL4 AD sequencing primer: 5'-TACCACTACAATGGAT-3'). The cDNA sequences were then BLAST-searched for gene homology with the GenBankTM databases at NCBI.
-32P]UTP
(PerkinElmer Life Sciences), 10 µM UTP, 0.5 mM ATP, 0.5 mM GTP, 0.5 mM CTP, 20 units of RNase inhibitor (Promega) and 50 units of T7 RNA polymerase.
RNA probes were purified on a 6% polyacrylamide-urea denaturing gel,
eluted in a 500 mM NH4CH3COO, 1 mM EDTA solution for 6 h at room temperature, ethanol
precipitated at
80 °C, and resuspended in water (500-1000
cps/µl) as previously described (16).
80 °C. Nuclear protein extracts were prepared from isolated nuclei as previously described (41). Protein concentration of cell extracts was determined by the Bio-Rad protein dye reagent.
80 °C with two intensifying screens.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (9K):
[in a new window]
Fig. 1.
Schematic description of human TTP cDNA
clones identified by yeast three-hybrid screening. Schematic
description of the TTP cDNA showing the relative positions of the
two zinc finger domains and proline repeats. Five clones homologous to
TTP were obtained following a yeast three-hybrid screen for PAI-2
AU-rich-binding proteins. The 5'-end points of the five TTP cDNA
clones obtained following this screen are indicated.
310 and
354 of the TTP
cDNA. All clones had the identical 3' structure, with the exception
of one clone (clone 5) that contained a 43-bp deletion at the terminus
of the 3'-UTR (Fig. 1). TTP contains 326 amino acids with three
tetraprolin repeats and two zinc fingers of the 2 Cys-Cys-Cys-His
class. All clones contained the two zinc finger domains and two of the
three tetraprolin domains suggesting the functional importance of these
domains for PAI-2 AU-rich RNA binding activity.
View larger version (34K):
[in a new window]
Fig. 2.
TTP binds to the PAI-2 ARE element.
Cytoplasmic extracts were prepared from mock-transfected HEK293 cells
or cells transiently transfected with 5 µg of plasmid pTTP-HA.
Extracts (4 ug) were incubated with 32P-labeled RNA probes
containing a single copy of the PAI-2 AU-rich element and subjected to
UV cross-linking. Replicate samples were digested with RNase-T1 either
alone (lanes 5-8) or in combination with RNase A
(lanes 9-12). All samples were then immunoprecipitated with
either rabbit preimmune serum (PI) or with rabbit anti-HA
antibodies (HA) in the presence of protein-A Sepharose beads
and subjected to SDS-PAGE. Gels were dried and autoradiographed. The
open arrow to the right of the figure shows the
HA-tagged TTP-RNA complex resistant to both RNase T1 and RNase A
digestion.
View larger version (44K):
[in a new window]
Fig. 3.
Overexpression of TTP destabilizes PAI-2
mRNA and reduces levels of PAI-2 protein in HEK293 cells.
HEK293 cells were transiently transfected with a constant amount of the
PAI-2 expression plasmid pCI-PAI-2, together with increasing amounts
(0-5.0 µg) of the TTP expression plasmid, pTTP-HA (pTTP).
After a 24-h transfection, RNA was extracted and probed for PAI-2 and
TTP mRNA by Northern blotting (panel A). Cytoplasmic
proteins were extracted from a replicate series of transfected cells,
and PAI-2 and TTP protein levels were assessed by Western blotting
(panel B). As shown in panel A, expression of
PAI-2 mRNA decreased with an increasing amount of TTP mRNA.
This was also reflected at the protein level (panel B) with
PAI-2 protein being reduced to undetectable levels with as little as
0.1 µg of plasmid pTTP-HA (lane 4). The lower
panel in A represents the ethidium bromide-stained gel
showing the relative levels of ribosomal RNA (rRNA). The lower
panel in B is a Coomassie Blue-stained SDS-PAGE gel
showing the equal loading of protein in all lanes.
View larger version (51K):
[in a new window]
Fig. 4.
The PAI-2 3'-UTR confers TTP-mediated
mRNA instability to -globin mRNA.
HEK293 cells were transiently co-transfected with a constant amount of
expression plasmid pCI-Glo-PAI-2 containing the PAI-2 3'-UTR fused to
the globin reporter transcript, together with increasing amounts
(0-5.0 µg) of the TTP expression plasmid, pTTP-HA (pTTP).
After a 24-h transfection, RNA was extracted and probed for globin and
TTP mRNA by Northern blotting. As shown in panel A
increasing levels of TTP mRNA correlated with a decrease in the
levels of the globin reporter transcript (panel A). The
arrow to the right of panel A
indicates the position of the stable truncated globin-PAI-2 mRNA
transcript formed in the presence of high levels of TTP. Similar
transient co-transfection experiments performed using the wild-type
globin expression plasmid with increasing amounts of pTTP-HA did not
reduce globin mRNA levels nor did it result in the formation of a
truncated transcript (panel B).
View larger version (56K):
[in a new window]
Fig. 5.
A single copy of the PAI-2 AU-rich element
partially confers TTP-mediated destabilization to
-globin. HEK293 cells were transiently
co-transfected with a constant amount (5 µg) of expression plasmid
pCI-Glo (lanes 1-5) or pCI-Glo-ARE (lanes 7-11)
or pCI-Glo-2ARE (lanes 13-17) together with increasing
amounts of plasmid, pTTP-HA (pTTP; 0-5.0 µg) as indicated
in the figure. After 24 h, RNA was extracted and probed for globin
and TTP mRNA by Northern blotting. As shown, a single copy of the
PAI-2 AU-rich element was capable of conferring
TTP-dependent instability to the globin reporter transcript
(compare lanes 1-5 with 7-11). Two copies of
the AU-rich element (2ARE) were even more effective than the
single copy (lanes 13-17) at destabilizing the globin
reporter transcript.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
animals were found to have high plasma levels of TNF protein while administration of anti-TNF antibodies largely reversed the phenotype, indicating that the inflammation was largely a consequence of the increase in the plasma levels of TNF. These findings therefore identified TTP as a component of a negative feedback loop that interferes with TNF production by destabilization of its mRNA. Since our study implicates TTP in the post-transcriptional regulation of PAI-2 gene expression, it would be predicted that
endogenous PAI-2 levels would also be elevated in TTP
/
due to the
loss of negative regulation, although any increase in PAI-2 expression that may be seen in these animals would need to be dissociated from the
possible effects of TNF, since this cytokine itself is a powerful
inducer of PAI-2 expression.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Dr. Andrew Clark for supplying the anti-TTP antibodies.
![]() |
FOOTNOTES |
---|
* This study was supported by grants obtained from the National Health and Medical Research Council of Australia (to R. L. M.).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.
These authors contributed equally to this work.
¶ To whom correspondence should be addressed: Monash University Dept. of Medicine, Box Hill Hospital, Box Hill 3128 Victoria, Australia. Tel.: 61-3-9895-0318; Fax: 61-3-9895-0332; E-mail: Robert.Medcalf@med.monash.edu.au.
Published, JBC Papers in Press, February 10, 2003, DOI 10.1074/jbc.M213027200
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ABBREVIATIONS |
---|
The abbreviations used are: PAI-2, plasminogen activator inhibitor type 2; TNF, tumor necrosis factor; IL, interleukin; ARE, AU-rich motif; UTR, untranslated region; HA, hemagglutinin; TTP, tristetraprolin; HEK, human embryonic kidney cells; GM-CSF, granulocyte-macrophage colony-stimulating factor.
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REFERENCES |
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