The plasminogen activator inhibitor 2 transcript is
destabilized via a multi-component 3¢ UTR localized
adenylate and uridylate-rich instability element in an
analogous manner to cytokines and oncogenes
Stan Stasinopoulos
1
, Mythily Mariasegaram
1
, Chris Gafforini
1
, Yoshikuni Nagamine
2
and Robert
L. Medcalf
1
1 Monash University, Australian Centre for Blood Diseases, Melbourne, Victoria, Australia
2 Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland
Introduction
The generation of the serine protease plasmin by the
plasminogen activator system is a critical event in a
variety of physiological processes, including fibrino-
lysis, development, wound healing and cell migration
[1–4]. Plasmin generation is regulated by two plasmin-
ogen activators: urokinase-type plasminogen activator
in the extracellular environment and tissue-type plas-
minogen activator in the circulation. The proteolytic
activities of both tissue-type plasminogen activator and
urokinase-type plasminogen activator are controlled by
plasminogen activator inhibitor types 1 and 2 (PAI-1
and PAI-2, respectively). One of the enigmatic features
of PAI-2 is that, although it can inhibit extracellular

in a manner analogous to some class I and II adenylate-uridylate elements
present in transcripts encoding oncogenes and cytokines. Hence, post-tran-
scriptional regulation of the PAI-2 mRNA transcript involves an interaction
between closely spaced adenylate-uridylate elements in a manner analogous
to the post-transcriptional regulation of oncogenes and cytokines.
Abbreviations
ARE, adenylate and uridylate rich element; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GM-CSF, granulocyte macrophage-
colony-stimulating factor; IL, interleukin; PAI-2, plasminogen activator inhibitor type 2; REMSA, RNA electrophoretic mobility shift assays;
RPA, RNase protection analysis.
FEBS Journal 277 (2010) 1331–1344 ª 2010 The Authors Journal compilation ª 2010 FEBS 1331
activator [5,6], it exists primarily as a nonglycosylated
intracellular protein. Over the past decade, evidence
has accumulated to suggest a role for PAI-2 in intra-
cellular events associated with apoptosis [7–11], prolif-
eration and differentiation [4,12], and the innate
immune response [7,13–15]. PAI-2 has also generated a
substantial level of interest because of its impressive
regulatory profile. It is one of the most responsive
genes known (i.e. it can be induced over 1000-fold),
and is regulated in a cell type-dependent manner by
phorbol esters [16,17], the phosphatase inhibitor, oka-
daic acid [18], tumour necrosis factor a [19,20], lipo-
polysaccharide [21,22] and elevated levels of serum
lipoprotein (a) [23]. Although there is a significant
transcriptional component to the regulation of PAI-2
expression by these agents, in recent years, the role of
post-transcriptional regulation has come to the fore
because a number of studies have shown that the half-
life of PAI-2 mRNA can also be altered in a treatment
and cell type-dependent manner [19,22,24–26].

of mammalian ARE-mediated mRNA decay can be
flexible. Recently, an excellent database compiling ARE
containing transcripts was established [46] and it has
been predicted that approximately 8% of human genes
code for transcript that contain AREs [47].
In a previous study, we defined the functional destabi-
lizing ARE element in the 3¢ UTR PAI-2 as a single
nonameric AU-rich sequence (UUAUUUAUU) located
304 nucleotides upstream of the poly(A) tail [24,48] and
suggested that tristetraprolin was a candidate PAI-2-
nonameric element binding protein involved in desta-
bilizing the PAI-2 mRNA transcript [49]. However,
subsequent work from our group demonstrated that
mutagenesis of the nonameric element only partially sta-
bilized the b-globin-PAI-2 3¢ UTR transcript [48], sug-
gesting the presence of additional functional
destabilizing regions within the PAI-2 3¢ UTR. In the
present study, we reveal that the nonameric ARE resides
within a 108 nucleotide U-rich (54%) region consisting
of three pentameric AU elements (one of which is a no-
nameric motif) and one atypical AU-rich region, and
that this extended region fully accounts for the complete
destabilizing activity of the PAI-2 3¢ UTR. Further-
more, functional mapping within the 108 AU-rich region
revealed that the essential destabilizing sequences, con-
sisting of the first two pentameric motifs and the atypical
AU-rich region, resided within a continuous 74 nucleo-
tide region, which we now define as the functional PAI-2
mRNA ARE element. The nonameric motif indeed com-
prises the core sequence that is essential for constitutive

a derivative (e.g. doxycycline) [50]. We cloned the full-
length wild-type PAI-2 3¢ UTR, and a mutant PAI-2
3¢ UTR containing a four nucleotide substitution within
the nonameric ARE (UUAUUUAUU to UUA
AAG
CUU) sequence into the unique BglII site in the b-globin
3¢ UTR of plasmid pTETGLO to create plasmids pTET
GLO
PAI)2
and pTETGLO
ARE II-MUT
, respectively.
These plasmids, including the empty vector, pTETGLO,
were transiently transfected into HT-1080 fibrosarcoma
TET-OFF cells and the decay characteristics (t
1 ⁄ 2
min)
of the various transcripts were determined after the addi-
tion of doxycycline by RNase protection analysis (RPA).
As shown in Fig. 1, the half-life of the wild-type b-globin
transcript was greater than 480 min, beyond the end
point of the experiment (based on the composite curve of
three separate experiments presented in Fig. 1), demon-
strating the high stability of this transcript. The half-life
of the b-globin
PAI)2
transcript was reduced to 158 min,
whereas the half-life of the b-globin
ARE II-MUT
transcript

3¢ UTRDARE
. This plasmid was transiently trans-
fected into HT1080 TET-OFF cells and the half-life of
the b-globin
ARED
transcript was shown to be
> 480 min (Fig. 3). Hence, this deletion resulted in
significant mRNA stabilization, with mRNA decay
kinetics reminiscent of the wild-type b-globin transcript
(Fig. 1). In addition, replacement of the 108 nucleotide
‘extended ARE’ with an equivalent length of an irrele-
vant sequence also substantially stabilized the tran-
script (data not shown) to an extent similar to that
seen previously with the b-globin and the b-globin
ARED
transcripts. Moreover, cloning the 108 nucleotide
‘extended ARE’ into the BglII site in the b-globin
3¢ UTR, creating plasmid pTETGLO
EXT.ARE
, resulted
A
B
C
Fig. 1. The PAI-2 3¢ UTR localized nonameric sequence only par-
tially contributes to PAI-2 mRNA instability. (A) Rabbit-b-globin-PAI-
23¢ UTR constructs prepared for the transient transfection of
HT1080-TET OFF cells. (B) HT1080-TET OFF cells were transfected
with the TET-responsive b-globin reporter plasmids described in (A).
After 16 h of incubation, doxycycline was added and total RNA was
isolated at the indicated times and analysed by RPA. The graph in

of these transcripts were 231 min for the b-globin
PAI)2
wild-type transcript, 204 min f or the b-globin
ARE I-MUT
,
193 min for the b-globin
ARE III-MUT
and 224 min
for the b-globin
ARE I+III-MUT
. These experiments dem-
onstrate that ARE I and III, both of which are
composed of classical pentameric sequence AUUUA,
do not independently contribute to the instability of the
PAI-2 transcript.
ARE I acts as a functional auxiliary element to
the core destabilizing ARE II site
To assess the possibility that the destabilizing activity
exhibited by the ‘extended ARE’ was the result of
A
B
C
Fig. 2. The PAI-2 3¢ UTR contains a 108 nucleotide functional ‘extended ARE’. A diagrammatic representation of the PAI-2 3¢ UTR showing
the location and sequence of the AU-rich regions of interest within the ‘extended ARE’, and the sequences of the various ‘extended ARE’
mutants that were generated.
Fig. 3. Deletion of the ‘extended’ ARE from the PAI-2 3¢ UTR
results in a stabilization reminiscent to the wild-type b-globin tran-
script. (A) Rabbit-b-globin-PAI-2 3¢ UTR constructs prepared for the
transient transfection of HT1080-TET OFF cells. Plasmids pTET-
GLO

(Fig. 3) transcripts, compared to the
wild-type b-globin
PAI)2
( 192 min) transcript in
this series of experiments (Fig. 6). This result suggests
that ARE I is an essential functional auxiliary element
to the core destabilizing ARE II sequence and that this
combination of AU-rich elements (ARE I⁄ ARE II)
plays a central role in determining the half-life of
the PAI-2 mRNA transcript under physiological
conditions.
Curiously, the half-life of the b-globin
ARE II+III
double mutant transcript was only partially stabilized
to 347 min (Fig. 6), which is also reminiscent of the
half-life of 333 min for the b-globin
ARE II-MUT
transcript (Fig. 1). This implies that ARE III is
unlikely to cooperate with the AREII ⁄ nonameric
element to contribute to the destabilizing activity
of the ‘extended ARE’ in the presence of an active
ARE I.
A
B
C
Fig. 4. The 108 nucleotide ‘extended ARE’ independently confers
mRNA instability in an analogous manner to the PAI-2 full-length
3¢ UTR. (A) Rabbit-b-globin-PAI-2 3¢ UTR constructs prepared for
the transient transfection of HT1080-TET OFF cells. Plasmids pTET-
GLO, Plasmids pTETGLO

ARE I+III-MUT
. (B) HT1080-TET OFF cells were transfected
with the TET-responsive b-globin reporter plasmids described in (A)
and the b-globin mRNA decay curves were quantified by RPA as
described in Fig. 1 and the Experimental procedures. The experi-
ments shown in (B) were repeated two or three times and each
point represents the mean ± SE.
S. Stasinopoulos et al. PAI-2 mRNA decay requires a multicomponent ARE
FEBS Journal 277 (2010) 1331–1344 ª 2010 The Authors Journal compilation ª 2010 FEBS 1335
The ‘extended ARE’ contains an alternate atypical
AU-rich auxiliary element that interacts with the
core ARE II sequence
Comparison of the human PAI-2 3¢ UTR with
those from a number of mammalian species (Fig. 7)
using clustalw [50a] analyses revealed a high degree
of conservation between the ARE II (nonameric)
sequences and a 12 nucleotide atypical AU-rich
sequence (labelled ARE IV) immediately 3¢ to ARE II.
To determine the extent to which this sequence
contributed to the decay rate, the same seven nucleo-
tide substitution (gUUAUUUAUUau
gcauuccuau) was
introduced into the abutting atypical ARE IV site
within the context of the full-length 3¢ UTR (Fig. 2)
to create the plasmid pTETGLO
ARE IV-MUT
.As
determined by our TET-regulated globin mRNA decay
system, disruption of this element resulted in a half-life
of the b-globin

ARE I+II-MUT
tran-
scripts (Figs 1, 3 and 6, respectively).
RNA electrophoretic mobility shift assays (REMSA)
were next performed to determine whether these adja-
cent ARE sites played a role in protein binding activ-
ity. Initial experiments confirmed that the extended
wild-type ARE sequence provided specific protein
binding sites for cytoplasmic proteins extracted from
HT1080 TET-OFF cells (Fig. S1A). Subsequent analy-
ses further indicate that mutations introduced into
ARE II substantially reduced protein binding activity,
which is consistent with our previous results using
shorter RNA probes [48]. However, mutations intro-
duced into the adjacent ARE IV had only a minimal
effect on binding activity. When both the ARE II and
IV sites were mutated simultaneously, binding activity
was reduced to the level seen with mutations in ARE
II alone (Fig. S1B). Hence, ARE IV does not appear
to modulate protein binding activity to the ‘extended
ARE’, despite the fact that it contributes to mRNA
stability. Whether this is a consequence of the limita-
tion of the REMSA approach or the influence of
alternative functional AREs (e.g. ARE I) remains
unknown.
Taken together, the results obtained in the present
study suggest that the functional PAI-2 3¢ UTR insta-
bility sequence consists of an essential core nonameric
sequence, for which the optimal destabilizing activity
A

ulated member of the plasminogen activator system,
and is one of the most highly inducible genes known.
Its expression can be dramatically increased in
response to cytokines, growth factors, hormones,
lipopolysaccharides and tumour promoters
[16,18,20,21,51]. Although the impressive induction of
PAI-2 has been attributed to transcriptional events,
work from the early to mid-1990s demonstrated that
PAI-2 gene expression could be regulated post-trans-
criptionally via the modulation of mRNA stability
[19,24].
We previously demonstrated that human PAI-2
mRNA was inherently unstable, with a half-life of
 1 h and that most of the destabilizing activity was
attributed to the 3¢ UTR [24] and, to a lesser extent,
an instability element within exon 4 of the coding
region [52]. It was originally predicted the nonameric
ARE (UUAUUUAUU) located 304 nucleotides
upstream of the poly(A) tail was largely responsible
for the 3 ¢ UTR driven-instability of the PAI-2 tran-
script. However, mutagenesis of this nonameric ARE
only partially stabilized both a HGH-PAI2-3¢ UTR
chimeric transcript [48] and a b-globin-PAI-2 3¢ UTR
chimeric transcript (present study). Work from other
groups has demonstrated that the presence of a single
nonameric element [UUAUUUA(U ⁄ A)(U ⁄ A)] within a
3¢ UTR has a modest effect on the stability of a repor-
ter transcript [34,53]; as such, we predicted that the
PAI-2 3¢ UTR contained additional functional instabil-
ity elements, AU-rich or otherwise [37,54,55], that

such, were functionally equivalent in an analogous
manner to the three pentameric motifs located in the
c-fos transcript [33]. However, this set of experiments
(Fig. 5) demonstrated that ARE I and III did not con-
tribute to transcript instability, either individually or in
combination (Fig. 5). We then sought an alternative
model to explain the destabilizing characteristics of the
PAI-2 ‘extended ARE’ element.
Fig. 7. CLUSTALW analyses of the PAI-2 ‘extended ARE’ from different mammalian species reveals a high degree conservation in the ARE II
and ARE IV regions; human (accession no. J02685), Pan troglodytes (accession no. XM_001148307), mouse (accession no. X16490), and rat
(accession no. X64563). The open boxes indicate the relative positions of the human ARE I, II, III and IV elements.
S. Stasinopoulos et al. PAI-2 mRNA decay requires a multicomponent ARE
FEBS Journal 277 (2010) 1331–1344 ª 2010 The Authors Journal compilation ª 2010 FEBS 1337
We next investigated the possibility that the struc-
ture of the PAI-2 ‘extended ARE’ was based on a
multidomain model consisting of an essential, func-
tional destabilizing core domain (e.g. the ARE II
nonameric sequence), for which the destabilizing activ-
ity was optimized by the presence of nearby auxiliary
AU-rich sequences. Figures 6 and 8 demonstrate that
the b-globin-PAI-2 3¢ UTR chimeric transcript was
only stabilized in an manner comparable to the b-glo-
bin and the b-globin
DARE
transcripts, upon the intro-
duction of two different sets of double mutations (e.g.
ARE II + ARE IV mutant and ARE II + ARE I
mutant). Taking into consideration the fact that muta-
genesis of either ARE I or ARE IV in isolation (Figs 5
and 8, respectively) did not influence the transcript’s

ARE IV cooperates with ARE II to destabilize mRNA
still remains unknown. The role of the ARE 1 site was
not investigated in the present study and will be the
subject of future research.
Functional multidomain ARE structures have been
observed in a variety of class I and II ARE elements,
including those of c-fos, GM-CSF and IL-8, amongst
others (Fig. 9) [28,29,33], and appear to function via
similar mechanisms. Of greatest relevance to the PAI-2
ARE is the c-fos multidomain class I ARE, for which
the structure and function has been characterized in
detail; this ARE is composed of two structurally dis-
tinct but functionally interdependent domains [33]
(Fig. 9). The c-fos ARE core sequence consists of three
pentameric motifs embedded within a U-rich region
and is independently capable of destabilizing a tran-
script. The c-fos ARE auxiliary domain II is a
20 nucleotide U-rich sequence that cannot indepen-
A
B
C
Fig. 8. An atypical AU-rich sequence (ARE IV) abutting 3¢ to the
ARE II pentameric sequence can optimize the mRNA destabilizing
activity of the ARE II nonameric sequence. (A) Rabbit-b-globin-PAI-2
3¢ UTR constructs prepared for the transient transfection of
HT1080-TET OFF cells. The AU-rich sequence (ARE IV) abutting 3¢
to the ARE II pentameric motif was mutated to create plasmid
pTETGLO
ARE IV-MUT
; a double ARE mutant that combined ARE

element (Figs 7 and 9) also behaves as a functional
auxiliary domain in the presence of a mutated ARE
IV (Fig. 8). Whether the two PAI-2 auxiliary domains
are simultaneously active cannot be determined from
the data obtained in the present study, although it
does remain a plausible hypothesis. However, we
suggest that, under normal physiological conditions,
the destabilizing activity of the core domain is prefer-
entially optimized by auxiliary domain I (Fig. 7, the
atypical AU-rich ARE IV; Fig. 9) based on the high
degree of homology in the equivalent sequences of
other species (Fig. 7). Moreover, the addition of the
second auxiliary sequence, domain I (Fig. 9), can sup-
port the destabilizing activity of the core domain in
the absence of domain IV (Fig. 8).
In summary, under normal physiological conditions,
the PAI-2 mRNA transcript is unstable, which we now
attribute to the presence of a multidomain AU-rich
element within the 3 ¢ UTR (Fig. 9). ARE-mediated
PAI-2 mRNA instability significantly contributes to
the low constitutive levels of PAI-2 protein; however,
the ARE can also modulate PAI-2 mRNA stability
during physiological conditions that require high levels
of PAI-2 gene expression and, subsequently, the contri-
bution of post-transcriptional regulation to PAI-2 gene
expression cannot be underestimated. The present
study has focused on the characterization and fine
mapping of the functional destabilizing AU-rich region
within the PAI-2 3¢ UTR under physiological condi-
tions that result in an unstable PAI-2 mRNA tran-

respectively.
Fig. 9. Multidomain structure of PAI-2 (accession no. J02685), c-fos (accession no. NM_005252), GM-CSF (accession no. M11220) and IL-8
(accession no. Y00787) AREs. Sequences of the AREs are shown with the AUUUAs underlined, and the relative positions of the core and
auxiliary domains are overlined.
S. Stasinopoulos et al. PAI-2 mRNA decay requires a multicomponent ARE
FEBS Journal 277 (2010) 1331–1344 ª 2010 The Authors Journal compilation ª 2010 FEBS 1339
Mutant variants of the PAI-2 3¢ UTR were generated via
overlap extension PCR mutagenesis [57] using SJS133 and
SJS134 as the external primers and the constructs pTET-
GLO
PAI)2
and pTETGLO
ARE II-MUT
as the templates.
Mutation of the ARE I-AUUUA and ARE III-AUUUA
sequences used primers SJS172 and SJS173, and SJS174 and
SJS175, respectively. Mutation of the atypical AU-rich
sequence used primers SJS259 and SJS260, and the creation
of the ARE II ⁄ ARE IV double mutant used primers SJS261
and SJS262. The mutagenesis of ARE I and III introduced
HindIII restriction sites and so the creation of the pTET-
GLO
3¢ UTRDARE
involved digesting construct pTET-
GLO
ARE I+III-MUT
mutant with HindIII to remove the
108 bp ARE, gel purifying the larger fragment and self-liga-
tion. The PAI-2 ‘extended ARE’ was amplified from plasmid
pTETGLO

the incubator for further incubation.
In vitro transcription and RNase protection assay
and northern hybridization
A cDNA library prepared with 1 lg of total RNA
extracted from an HT1080 TET-off cell line transiently
transfected with pTETBBB was used to generate the rabbit
b-globin and glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) riboprobes. Accordingly, a 295 bp b-globin frag-
ment that spans the first intron was amplified using primers
SJS167 and SJS170 and a 155 bp GAPDH fragment was
amplified using primers ALS030 and SJS209; these frag-
ments were then cloned into the pGEM-T Easy vector
(Promega, Madison, WI, USA) generating pTEasy-Globin
and pTEasy-GAPDH. For in vitro transcription, 500 ng of
SpeI linearized pTEasy-Globin and SacII linearized
pTEasy-GAPDH were incubated for 1 h in the presence of
50 lCi [a-
32
P]UTP (PerkinElmer Life and Analytical Sci-
ences, Inc., Waltham, MA, USA), 10 lm UTP, 0.5 mm
ATP, 0.5 mm CTP, 0.5 mm GTP, 40 U of RNase Inhibitor
(Promega Corporation, Madison, WI, USA) and either
Table 1. PCR and overlap PCR mutagenesis primers. The name, nucleotide sequence, orientation and GenBank nucleotide reference (where
available) are provided. The introduced mutations are underlined,the restriction enzyme sites are italicized, and lower case indicates the T7
promoter sequence. PAI-2 cDNA (accession no. M18082), GADPH cDNA (accession no. M33197), pTETBBB plasmid sequence from Profes-
sor A. B. Shyu (University of Texas Medical School, Houston, TX, USA). nt, nucleotide.
Primer Nucleotide sequence (5¢-to3¢) Orientation
SJS133 CGGA
AGATCT
AACTAAGCGTGCTGCTTC Forward (nt 1281–1298 PAI-2)

SJS276 GtaatacgactcactataGGGATCATGCCCATTTAG T7Forward (nt 1491–1508 PAI-2)
PAI-2 mRNA decay requires a multicomponent ARE S. Stasinopoulos et al.
1340 FEBS Journal 277 (2010) 1331–1344 ª 2010 The Authors Journal compilation ª 2010 FEBS
50 U of T7 Polymerase for pTEasy-Globin or 50 U of SP6
polymerase for pTEasy-GAPDH. The radioactive products
were purified according to the RPA III kit instructions
(Ambion Inc., Austin, TX, USA).
The RNase protection assay was carried out using the
RPA III kit (Ambion) according to the manufacturers
instructions with 7.5 lg of total RNA isolated from the
transiently transfected HT1080 TET-OFF cells. The prod-
ucts were resolved on a denaturing gel (7 m urea ⁄ 5%
PAGE) and visualized by autoradiography. Signals were
quantified using the ImageQuant, version 5 (Amersham
Biosciences, Piscataway, NJ, USA) and the results were
presented in graphical form after correcting for variations
in GAPDH levels between time point samples.
Total RNA (10 lg) was resolved on a 1% formaldehyde-
agarose gel and transferred to a Hybond-N nylon mem-
brane (GE Healthcare, Piscataway, NJ, USA). RNA blots
were stained with methylene blue to confirm for equal load-
ing and transfer. Hybridization was performed by the
Rapid-Hyb hybridization protocol (GE Healthcare) using
random primed [a-
32
P]dATP-labelled cDNA probes corre-
sponding to rabbit b-globin and human GAPDH isolated
from plasmids pTEasy-Globin and pTEasy-GAPDH.
Hybridization signals were visualized and quantified with
a PhosphorImager (Molecular Dynamics, Sunnyvale, CA,

generate the various DNA fragments from plasmids pTET-
GLO
PAI)2
, pTETGLO
ARE II-MUT
, pTETGLO
ARE IV-MUT
and pTETGLO
ARE II+IV-MUT
. The unrelated 114 nucleo-
tide RNA probe was derived from T7 transcribed KpnI
digested pBluescript KS+. The RNA probes were purified
on a 6% polyacrylamide-urea gel, eluted in a 500 mm
NH
4
CH
3
COO, 1 mm EDTA solution overnight at room
temperature, ethanol precipitated at –80 °C and resus-
pended in water (30 000 c.p.s.ÆlL
)1
). To prepare extracts
for REMSAs, cells (80% confluence) were collected by
trypsinization, washed with NaCl ⁄ P
i
, then lysed for 10 min
on ice in 300 lL of cytoplasmic extraction buffer (10 mm
Hepes, pH 7.1, 3 mm MgCl
2
,14mm KCl, 0.2% NP-40,

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Supporting information
The following supplementary material is available:
Fig. S1. The ‘extended ARE’ provides specific binding
sites for cytoplasmic proteins.
This supplementary material can be found in the
online version of this article.
Please note: As a service to our authors and readers,
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should be addressed to the authors.
PAI-2 mRNA decay requires a multicomponent ARE S. Stasinopoulos et al.
1344 FEBS Journal 277 (2010) 1331–1344 ª 2010 The Authors Journal compilation ª 2010 FEBS