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INTRODUCTION |
Tumor necrosis factor-
(TNF-
)1 is a cytokine
predominantly produced by macrophages but also by lymphocytes, NK
cells, astrocytes, and other cell types. The most powerful inducers of
TNF-
production by macrophages are the lipopolysaccharides (LPS),
which are membrane components released by Gram-negative bacteria in the
course of infection (1). It is now well established that the induction of TNF-
production upon stimulation of macrophages by LPS results from both an enhancement of TNF-
gene transcription and a
translational derepression of the mRNA. In unstimulated
macrophages, TNF-
mRNA translation is blocked. Upon stimulation
with LPS, this repression is overcome, and TNF-
mRNA becomes
efficiently translated (2). The key element involved in this regulation
is the AU-rich element (ARE) located in the 3'-untranslated region
(-UTR) of TNF-
mRNA (3). This 70-nucleotide-long sequence is
composed of several repeats of the AUUUA pentamer. The physiological
importance of TNF-
mRNA translational control is demonstrated by
the fact that the expression of a TNF-
transgene lacking its 3'-UTR
in mouse leads to severe inflammatory disorders (4).
Similar AREs are found in the 3'-UTR of a growing number of mRNAs
encoding cytokines, protooncogenes, or other transiently expressed
proteins (5). These sequences have also been shown to regulate mRNA
stability (6).
In former studies, we reported that TNF-
mRNA ARE can form two
complexes with proteins present in cytosolic macrophage extracts. One
of these complexes (complex 1) forms with extracts of both unstimulated
and LPS-stimulated macrophages and requires a large fragment of the ARE
containing clustered AUUUA pentamers. The other complex (complex 2) is
only detected after cell activation, binds to a minimal UUAUUUAUU
nonamer, and is composed of a 55-kDa protein (7, 8).
To identify the proteins involved in both complexes, we designed a
cloning strategy based on the differential screening of a macrophage
cDNA expression library with TNF-
mRNA 3'-UTR riboprobes containing or not the ARE. By this method, we isolated the cDNA encoding the short 40-kDa isoform of the RNA-binding protein TIAR. We
show that TIAR specifically binds TNF-
ARE and corresponds to the
protein involved in the formation of complex 1. Moreover, analysis of
TIAR subcellular localization by immunostaining reveals that TIAR is
mainly found in the cytoplasm of murine macrophages.
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EXPERIMENTAL PROCEDURES |
Materials--
Enzymes used in this study were purchased from
Boehringer Mannheim and Life Technologies Inc. LPS (Escherichia
coli strain 0.127.B8), diethyl pyrocarbonate, and anti-actin
antibody were obtained from Sigma.
Isopropyl-1-thio-b-D-galactopyranoside and oligonucleotides
were purchased from Life Technologies Inc. Lysozyme was purchased from
Appligene Oncor. DNase I and polyC were purchased from Amersham
Pharmacia Biotech.
Goat anti-TIAR polyclonal antibody directed against a C-terminal
peptide of TIAR and rabbit anti-NF-
B antibody were purchased from
Santa Cruz. The goat IgG control antibody was purchased from Rockland
(Gilbertsville, Pa). Mouse actin cDNA cloned in the pBluescript SK(
) phagemid was purchased from Stratagene.
Cell Culture--
RAW 264.7 mouse macrophages were maintained as
described previously (7). LPS was added at a final concentration of 10 ng/ml for 2 h in all experiments.
Expression Library Screening--
A RAW 264.7 mouse macrophage
cDNA library was prepared according to a previously described
method (5) and was inserted into the pUC-19 vector within the
PstI and BamHI restriction sites. The library or
the pGEX3X-37CR plasmid-expressing AUF1 (generously provided by Dr.
Gary Brewer, Bowman Gray School of Medicine, Wake Forest University,
Winston Salem, North Carolina) or the pGEX5X-1-actin plasmid-expressing
actin (a gift from V. Dilbeck, University of Brussels) were
electroporated into MC1061 bacteria and plated to obtain 10,000 colonies/dish (dish diameter: 13.5 cm). After overnight incubation, 1 replicate/dish was performed on nitrocellulose filters (Schleicher & Schuell). These "master" replicates were used to perform secondary
(2 to 5) replicates. The master and the secondary replicates were then
placed in dishes containing ampicillin (100 µg/ml) or ampicillin (100 µg/ml) and isopropyl-1-thio-b-D-galactopyranoside (1 mM), respectively, and incubated overnight at 37 °C. The
master replicates were subsequently stored at 4 °C. The secondary
replicates were hung up in a sealed tube containing a 1-cm layer of
pure chloroform for 35 min. Filters were then transferred into plastic bags (6 filters/bag maximum) and soaked in 300 ml of autoclaved washing
buffer (50 mM Tris, pH 8, 150 mM NaCl, 5 mM MgCl2, 1/1000 (v/v) diethyl pyrocarbonate )
containing lysozyme (1 mg/l) and DNase I (1 mg/l) for 2 h at room
temperature with light shaking. The washing buffer was then replaced by
300 ml of fresh washing buffer, and the incubation was prolonged for 4 additional h. The washing buffer was replaced for a second time, and
the filters were soaked overnight. The filters were then rinsed three
times for 5, 30, and 60 min, respectively, in 300 ml of binding buffer (40 mM Tris, pH 8, 4 mM EDTA, 200 mM NaCl, 3.6 mM 2-mercaptoethanol, 1/1000 (v/v)
diethyl pyrocarbonate) at room temperature with light shaking. For the
binding with the RNA probe, each membrane was transferred into a
plastic bag containing 35 ml of binding buffer and incubated for 10 min
at room temperature. Heparin (0.55 mg/ml) was added, and a subsequent
incubation of 10 min was performed. PolyC (10 µg/ml) was then added,
and the membranes were incubated for an additional 30 min. Finally,
100 × 106 cpm of riboprobe was added per bag, and the
bags were incubated overnight at room temperature with light shaking.
The membranes were washed twice for 10 min in 100 ml of binding buffer
at room temperature with light shaking, dried for 15 min on 3MM paper, and autoradiographed.
In Vitro Transcription--
The DNA constructs used in this
study were previously described (7). The riboprobes used for the
screening procedure and electrophoretic shift assay (EMSA) were
synthesized with the transcription kit purchased from Epicentre
(Madison, WI) according to the following method. Briefly, to generate
100 × 106 cpm (approximately 1.5 ×108
cpm/µg), 4 µl of SP6 transcription buffer 5×, 2 µl of 100 mM dithiothreitol, 1 µl of 10 mM ATP, 1 µl
of 10 mM CTP, 1 µl of 10 mM GTP, 3 µl of 1 mM UTP, 80 µCi of [
-32P]UTP (800 Ci/mmol), 3 µl of linearized DNA (0.5 µg/µl), and 1 µl of SP6
RNA polymerase were mixed and incubated for 2 h at 37 °C. The
transcription reaction was then treated with DNase for 15 min, brought
to a volume of 50 µl with H2O, and extracted with phenol/chloroform. The riboprobe was then purified on a P10 minicolumn, and the volume was increased to 300 µl. The probes were stored at
70 °C.
DNA Sequencing--
DNA sequencing was performed using the
Thermo Sequenase radiolabeled terminator cycle sequencing kit (Amersham
Pharmacia Biotech).
EMSA--
S100 macrophage extracts and EMSA were carried out
exactly as described previously (7). Supershifts with anti-TIAR or
control antibodies were performed by incubating 15 µg of S100
macrophage extract with 0.2 µg of anti-TIAR antibodies or control
antibodies for 25 min on ice in a total volume of 15 µl before the
EMSA. The EMSA were electrophoresed on nondenaturing 3.5%
polyacrylamide gels.
Immunodepletion of Macrophage Extract before EMSA--
150 µg
of S100 extract from uninduced RAW macrophages were incubated with 4 µg of anti-TIAR antibody or control IgG in the presence of 50 µl of
protein G-agarose beads (Santa Cruz, Ca.) for 16 h on a rotating
wheel at 4 °C. The samples were centrifuged for 30 s, and the
supernatant was incubated with fresh antibodies and protein G beads for
24 h. After elimination of the beads by centrifugation, the
protein concentration of the samples was determined by using a BCA kit
(Pierce), and 15 µg of each samples were used in an EMSA as described previously.
Northern Blot Analysis--
Cytoplasmic RNA was isolated from
RAW 264.7 cells according to the method previously described. Northern
blot analysis was performed as described in Sambrook et al.
(9) using 10 µg of total RNA. The blot was hybridized with a TIAR
cDNA probe synthesized with the Rediprime kit (Amersham Pharmacia
Biotech). The same blot was subsequently hybridized with an actin probe
to normalize the amount of RNA loaded on the gel.
Western Blot Analysis--
Protein extracts were prepared by
recovering the cells in 0.25 M Tris, pH 7.8, and were lysed
by three cycles of freezing and thawing. The protein concentration in
the extracts was determined by the BCA protein assay (Bio-Rad).
Fifteen µg of each extract were run on a 10% SDS-polyacrylamide gel,
and the Western blot was performed as described elsewhere (10). TIAR
protein and actin were immunodetected using anti-TIAR and anti-actin
antibodies, respectively.
Immunoperoxydase Cell Staining--
Briefly, 105
cells were centrifuged on slides, fixed with methanol for 5 min at
20 °C, and stored at room temperature until use. The slides were
rinsed in phosphate-buffered saline, incubated overnight with the
primary antibody (anti-TIAR, anti-NF-
B, control IgG) (10 µg/ml) at
4 °C in a humidified chamber, and rinsed again in phosphate-buffered
saline. Slides were subsequently incubated for 1 h in
phosphate-buffered saline in the presence of the secondary antibody
coupled to peroxydase (20 µg/ml). Peroxydase activity was revealed by
using the AEC (3-amino-9-ethyl-carbazol) method (Sigma). The slides
were mounted in aquatex (Merck) and observed at 100× magnification.
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RESULTS |
Expression Library Screening for TNF-
ARE-binding
Protein--
TNF-
ARE sequence was previously shown to form two
different complexes with proteins of macrophage S100 extracts. These
two complexes differ in their electrophoretic mobilities and in their recognition motifs within the ARE. Moreover, although the complex of
low electrophoretic mobility (complex 1) can be detected with S100
extracts from both unstimulated and LPS-stimulated macrophages, the
other complex (complex 2) is formed only upon LPS stimulation (7, 8).
To clone the cDNAs encoding the proteins involved in these
complexes, we developed an expression library screening method. We set
up this method with a plasmid encoding AUF1, which has been shown to
bind to AREs derived from several mRNAs (11). As negative control,
we used a plasmid encoding actin. Bacteria expressing either AUF1 or
actin were plated and replicated to perform a binding with TNF-
mRNA 3'-UTR riboprobes containing or not containing the ARE (Fig.
1 and "Experimental Procedures"). Although TNF 3'-UTR riboprobe bound to the replicate of bacteria encoding AUF1, no signal could be detected with bacteria expressing actin. The binding of TNF 3'-UTR riboprobe to AUF1-expressing colonies
involves the ARE, because no signal was detectable with the TNF
3'-UTR
AU riboprobe (Fig. 2).

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Fig. 1.
Schematic representation of the plasmid
templates used for in vitro transcription of the RNA
probes. Numbers indicate nucleotide positions within
the 3'-UTR of TNF mRNA using the third nucleotide of the stop codon
as reference 0. (AU)n represents the AU-rich element. The
exact sequence of this element is indicated, and the consensus
pentanucleotide motifs are underlined.
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Fig. 2.
Validation of the screening method with AUF-1
and actin expression vectors. E. coli strain MC1061 was
transformed either with AUF-1 or actin-expression vectors and plated at
10,000 colonies/dish, replicated on nitrocellulose membranes, and
incubated with the 3'TNF or 3'TNF AU RNA probes as described under
"Experimental Procedures."
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We then screened the RAW 264.7 macrophage cDNA library in the same
conditions to identify clones encoding proteins specifically binding
TNF ARE. In a first step, clones detected by the binding of TNF 3'-UTR
riboprobe were isolated and replated at high density. Two replicates of
the resulting plates were prepared as described previously and
submitted to a second differential screening with 3'TNF and 3'TNF
AU
probes. As illustrated in Fig. 3, one of
the clones detected by the first screening specifically bound to 3'TNF probe in the secondary differential screening. The cDNA of five independent colonies detected with the 3'TNF probe were sequenced, and
all were encoding the 40-kDa isoform of the RNA-binding protein TIAR.

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Fig. 3.
Identification by the expression screening
method of TIAR as a protein specifically binding to the TNF ARE.
The RAW cDNA library was screened as indicated under
"Experimental Procedures." Positive clones were isolated from the
master plates, diluted in LB medium, replated, and engaged in a
secondary differential screening using the 3'TNF or 3'TNF AU probes
(equal amount of cpm for each probe). The figure is representative of
three independent experiments of differential screening of the clone
encoding TIARb.
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TIAR Is Involved in the Formation of Complex 1--
To determine
whether TIAR is involved in one of the two complexes that can form with
TNF ARE, we analyzed the binding ability of TIAR to the 3'd2 probe
(Fig. 1), which has been previously shown to selectively form complex 2 and not complex 1 (Ref. 8 and Fig.
4A). As illustrated in Fig.
4B, the 3'd2 riboprobe binds very weakly to TIAR-expressing
colonies, indicating that TIAR might not be involved in complex 2. We
next performed EMSAs with TNF 3'-UTR riboprobe and macrophage extracts
in the presence of anti-TIAR or control IgG antibodies. Fig.
5A shows that the addition of
anti-TIAR antibody in the EMSA markedly alters the electrophoretic mobility of complex 1 in comparison with a control IgG. On the other
hand, complex 2 migration is not affected by anti-TIAR antibody, further confirming that this complex does not involve TIAR. To corroborate the involvement of TIAR in complex 1, we performed EMSAs
with macrophage extract depleted of TIAR by immunoprecipitation with
anti-TIAR antibody (expression library screening method). A significant
decrease in complex 1 intensity was observed as compared with the level
obtained when the immunodepletion was performed with a control IgG
(Fig. 5B). In parallel, we verified by Western blot analysis
that immunodepletion of the extract by anti-TIAR antibody correlated
with an increase of TIAR present in the immunoprecipitated protein
G-Sepharose pellet (data not shown).

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Fig. 4.
Comparison of the formation of complexes 1 and 2 and the binding specificity of TIAR to TNF ARE.
A, EMSA performed with cytosolic (cyto.) extract
of RAW cells induced with LPS (10 ng/ml, 2 h) and 3'TNF or 3'd2
RNA probe. B, comparison of the binding specificity of TIARb
to the 3'TNF and 3'd2 probes. The probes were labeled with
[32P]UTP, and an equal amount of cpm for each probe was
used in the binding reactions.
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Fig. 5.
Identification of TIAR as a component of
complex 1. A, supershift of complex (Compl.)
1 by anti-TIAR antibody. EMSA experiment was performed with cytosolic
extract from noninduced (NI) or LPS-activated RAW cells and
the 3'TNF probe in the presence of anti-TIAR antibody or control IgG.
The figure is representative of four independent experiments.
B, EMSA was performed with macrophage extract immunodepleted
with either an anti-TIAR antibody or a control IgG (expression library
screening method). The figure is representative of two independent
experiments.
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TIAR Expression in Macrophages--
tiar has been previously
described as a widely expressed gene at least at the mRNA level.
Moreover, tiar gene can be expressed into two isoforms of 40 and 42 kDa (TIARb and TIARa, respectively), resulting from alternative
splicing of the precursor mRNA (12). TIARa contains an additional
17-amino acid stretch in the first of the three RNA recognition motifs.
Because of its location in a loop of the first RNA recognition motif,
this 17-amino acid peptide has been suggested to be important for the
protein RNA binding specificity (13). We characterized the expression
of tiar gene in unstimulated and LPS-stimulated macrophages
at the RNA and protein levels. The Northern blot analysis of TIAR
mRNA accumulation shows that TIAR mRNA is equally expressed as
a 1.6-kilobase transcript in both unstimulated and LPS-stimulated RAW
cells (Fig. 6A). The
resolution of the gel electrophoresis did not allow the separation of
the mRNA species encoding the two isoforms. At the protein level,
we observed that the 40-kDa TIARb is significantly more expressed than
the 42-kDa TIARa. As at the RNA level, TIAR proteins are equally
expressed in unstimulated and LPS-stimulated macrophages (Fig.
6B).

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Fig. 6.
Analysis of TIAR expression in RAW
macrophages. A, Northern blot analysis of total RNA
from noninduced (NI) or LPS-activated RAW cells using a TIAR
or actin cDNA probes. The size of the corresponding mRNAs is
indicated. B, Western blot analysis of total protein extract
from noninduced or LPS-activated RAW cells using an anti-TIAR or
anti-actin antibody. The molecular mass of the proteins is indicated.
The figure is representative of three independent experiments.
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Subcellular Localization of TIAR in Macrophages--
TIAR, which
is a protein related to TIA-1, has been initially described to trigger
DNA fragmentation in permeabilized thymocytes (14) and to be localized
mainly in the nucleus. However, upon Fas-mediated apoptosis, TIAR has
been shown to be translocated to the cytoplasm (15). Because we
identified TIAR as a component of complex 1, which is formed upon
incubation of TNF ARE with macrophage S100 cytosolic extracts, we
determined TIAR subcellular localization in macrophages by an
immunostaining analysis. This experiment showed that similarly to
NF-
B, TIAR is predominantly found in the cytoplasm of RAW
macrophages (Fig. 7). This cytoplasmic localization of TIAR correlates with its ability to form complex 1 from
S100 cytosolic extract.

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Fig. 7.
Subcellular localization of TIAR in RAW
macrophages. Slides on which RAW macrophages were fixed as
described under "Experimental Procedures" were incubated with an
anti-TIAR antibody or an anti-NF- B antibody or a control IgG and
stained by the immunoperoxydase AEC (3-amino-9-ethyl-carbazol) method.
This experiment was performed three times, and the figure illustrates a
representative field of each staining.
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DISCUSSION |
It is now well established that AREs play a major role in the
post-transcriptional regulation of several transiently expressed genes.
These elements have been classified in two main categories according to
the number and the distribution of AUUUA pentamer repeats. Class I AREs
contain one to three copies of the AUUUA pentamer in a U-rich context.
Prototypes of these class I AREs are found in the 3'-UTR of c-Myc and
c-Fos mRNAs and confer mRNA instability. Class II AREs are
defined by the presence of multiple clustered pentamers and are
preferentially found in the 3'-UTR of cytokine mRNAs (16).
Depending on the systems considered, these AREs mediate mRNA
instability and/or translational blockade. Several proteins have been
reported to bind AREs (reviewed in Refs. 17 and 18). However, based on
their molecular mass and sequence affinity, none of these factors
seemed to correspond to the proteins involved in the complexes formed
with TNF ARE that we previously described. The expression library
screening method with RNA probes has been successfully used to clone
cDNAs of proteins specifically binding to RNA through different
types of RNA binding domains (19, 20). Therefore, we developed a similar but simplified strategy based on the direct screening of
bacterial colonies transformed with cDNA-expressing plasmids. This
method led to the identification of TIARb as a protein specifically binding TNF ARE. Moreover, we have confirmed by supershift experiments that TIAR corresponds to the constitutive TNF ARE-binding protein present in macrophage cytosolic extract previously described as complex
1. Indeed, EMSA performed in the presence of anti-TIAR antibody
markedly alters the electrophoretic mobility of complex 1. However, it
should be noted that the band corresponding to complex 1 is not totally
supershifted and that increasing the amount of anti-TIAR antibody does
not modify the supershift ratio (data not shown). This might result
from a limited affinity of anti-TIAR antibody. Alternatively, the
remaining band could correspond to another complex co-migrating with
complex 1. The supershift of complex 1 by anti-TIAR antibody together
with the observation that complex 1 decreases upon immunodepletion of
TIAR from macrophage extract indicates that this complex contains
either one or both TIAR isoforms. The screening of 40,000 clones of the
RAW 264.7 cDNA library led to the isolation of TIARb and not of
TIARa, suggesting that TIARb is the isoform involved in complex 1. However, the predominant expression of TIARb in macrophages could also
explain the preferential detection of the TIARb isoform by the
screening procedure.
We have shown by an immunostaining analysis that TIAR is essentially
cytoplasmic in murine macrophages. This result correlates with the
identification of TIAR as a protein binding TNF ARE present in S100
cytosolic extract. TIAR has been previously cloned on the basis of its
homology to TIA-1, a protein mainly expressed in T lymphocytes and
shown to be able to trigger DNA fragmentation in
digitonin-permeabilized thymocytes (21). Taupin et al. (15) have also shown that TIAR is mainly located in the nucleus of human
Jurkat 77 cells and relocates to the cytoplasm of these cells during
Fas-mediated apoptosis. At this point, the difference observed in the
localization of TIAR in human T lymphocytes and murine macrophages is
not explained. TIAR localization might be cell
type-dependent, or TIAR subcellular distribution could be correlated with different functional activities of this protein. However, we cannot exclude that a variation in the epitopes recognized by the antibodies used to determined TIAR localization could account for the difference observed. Indeed the monoclonal antibody used to
study TIAR relocalization in FAS-activated Jurkat cells binds an
epitope located in the highly conserved second RNA recognition motif of TIAR and recognized a 50-kDa polypeptide in Western blot. This
50-kDa protein is not detected in Western blot experiments performed
with RAW 264.7 cell extract and an anti-TIAR antibody directed against
a C-terminal peptide of TIAR ( Fig. 6 and "Experimental Procedures").
Although TIAR was known as a RNA-binding protein (13), this study
identifies the first RNA regulatory sequence recruiting TIAR. We have
previously reported that a cluster of five overlapping AUUUA pentamers
is the minimal sequence within TNF ARE required for complex 1 formation. We have also shown that complex 1 can form with TNF and
granulocyte-macrophage colony-stimulating factor class II AREs and not
with c-myc class I ARE. Therefore, TIAR being a component of
complex 1 can be considered as a class II ARE-specific binding protein.
TNF mRNA is both unstable and translationally regulated in
macrophages (2, 22). Recently, tristetrapolin, a CCCH zinc finger
protein, has been reported to mediate TNF mRNA destabilization in
macrophage by interacting with TNF mRNA ARE (18), but the proteins
involved in the translational control of TNF mRNA are not
identified yet. As TIAR is located in the cytoplasm of macrophages and
binds to TNF ARE independently of LPS treatment of the cells, it could
mediate TNF translational repression. Activation of macrophages with
LPS leads to the release of the translational blockade and is
accompanied by the binding of a 55-kDa protein on TNF ARE, which could
be responsible for this effect (8).
The identification of the 55-kDa protein along with further
characterization of TIAR function in macrophage will provide more insight into the mechanism of cytokine mRNA translational control mediated by AREs.