From INSERM U386, Université Victor Segalen,
Institut de Formation et de Recherche Pathologies Infectieuses, 146 rue
Léo Saignat, 33076 Bordeaux, France and § Laboratoire
de Chimie Biologique, Université Libre de Bruxelles, 67 rue des
chevaux, 1640 Rhode St Genèse, Belgium
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In monocyte/macrophage cells, the translation of
tumor necrosis factor- (TNF-
) mRNA is tightly controlled. In
unstimulated cells, TNF-
mRNA is translationally repressed.
However, upon stimulation of the cells with various agents
(e.g. lipopolysaccharides (LPS) and viruses), this
repression is overcome and translation occurs. The key element in this
regulation is the AU-rich sequence present in the 3'-untranslated
region of TNF-
mRNA. Several groups have described the binding
of proteins on AU-rich elements (AREs). We have previously reported the
binding of two cytosolic protein complexes (1 and 2) to the TNF-
mRNA ARE, one of which (complex 2) is observed only following
induction of TNF-
production by LPS. In this report, we have
demonstrated that complex 1 involves a long fragment of the ARE,
whereas the formation of the LPS-inducible complex 2 requires a
minimal sequence which corresponds to the nonanucleotide UUAUUUAUU.
Furthermore, we show that the RNA-binding protein involved in complex 2 has an apparent molecular mass of 55 kDa. Finally, we tested other AREs
for their ability to form complex 2. We observed that the ARE derived
from granulocyte/macrophage colony-stimulating factor mRNA, which
does contain the nonanucleotide, is able to sustain the LPS-induced
binding of the 55-kDa protein. However, c-myc mRNA,
which does not contain the nonanucleotide, is unable to promote the
formation of any LPS-induced complex.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Translational control is now considered to be an important means of gene regulation. The expression of many mRNAs, including those encoding the p53 protein (1), lipoxygenase (2), and cytokines (3-5), has been shown to be controlled at a translational level. In each of these cases, the sequences responsible for this effect have been mapped to the 3'-untranslated regions (UTRs)1 of the genes.
AU-rich elements (AREs) are found in the 3'-UTRs of many oncogene and cytokine mRNAs (6). Originally, these motifs, in particular the AUUUA pentanucleotide, were shown to destabilize messages when placed downstream of the coding sequence (7). ARE-containing mRNAs have been divided into two classes based on the number and distribution of the AUUUA pentamer. Oncogene mRNAs generally fall into class 1, having one to three AUUUA sequences spaced throughout the 3'-untranslated region. The poly(A) tails of these mRNAs are degraded synchronously. Class 2 is mainly composed of cytokine mRNAs, and is defined by the presence of multiple clustered pentamers. This confers a processive mechanism of poly(A) tail decay (8).
AREs in cytokine mRNAs are also implicated in translational control
(3, 4, 9). Tumor necrosis factor- (TNF-
) is not expressed in most
cell types or in unstimulated monocyte-derived cell lines. Using a
reporter gene linked to a series of deletions of the TNF-
3'-UTR,
the sequences mediating this repression were localized to an ARE. In
macrophages, TNF-
production is rapidly induced following
stimulation with lipopolysaccharide (LPS), a component of the cell wall
of Gram-negative bacteria. Although part of this induction can be
accounted for by an increase in both the transcription of the TNF-
gene and the stability of the mRNA, it has been shown that the most
important event following LPS stimulation is a relief from the
ARE-mediated translational blockade (4).
Recently, we and others have shown that several proteins can complex
with the ARE of the TNF- mRNA in unstimulated cells (10, 11).
Furthermore, an additional protein complex is recruited to this region
following cell activation, suggesting a role in TNF-
mRNA
translational derepression (10). Here, we show that this inducible
complex contains a previously undescribed protein of 55 kDa and
demonstrate that it binds to a single UUAUUUAUU sequence, but not to
the AUUUA pentanucleotide.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials-- Enzymes used for construction of the different plasmids were purchased from Promega or Life Technologies, Inc. RNase T1 was purchased from Boehringer Mannheim. RNase A and LPS (Escherichia coli strain 0.127:B8) were obtained from Sigma. Synthetic RNAs were purchased from Eurogentec (Belgium).
Cell Culture-- RAW 264.7 mouse macrophages were maintained as described previously (10). LPS was added at a final concentration of 10 ng/ml for 2 h in all experiments.
DNA Constructs--
The TNF- ARE probes have been previously
described (10). The GM-CSF ARE probe was synthesized by inserting the
227-base pair NcoI-BglII fragment corresponding
to the distal part of the GM-CSF mRNA 3'-UTR (3) into the SP64
plasmid previously cut by PstI, blunted, and cut by
BamHI. The c-myc 3'-UTR construct was a kind gift
of Dr. Blanchard, Montpellier, France.
In Vitro Transcription--
To synthesize riboprobes, the
pBluescript derived plasmids were linearized with XbaI and
transcribed using T7 RNA polymerase (Life Technologies, Inc.); the
TNF- ARE and GM-CSF plasmids were linearized with EcoRI
and transcribed using SP6 polymerase (Life Technologies, Inc.), and the
c-myc plasmid was linearized with XhoI and
transcribed using T3 polymerase (Life Technologies, Inc.). All
reactions were carried out as described previously (10) for
electrophoretic mobility shift assay (EMSA) probes, and with [
-32P]UTP (100 µCi; 3000 Ci/mmol) for the UV
cross-linking probes. Unlabeled RNAs were synthesized using 0.5 mM amounts of each nucleotide.
Treatment of TNF- ARE with RNase T1--
TNF-
ARE probe
was digested with RNase T1, which cleaves at the 3' side of G residues,
yielding three major fragments of 39, 20, and 10 nucleotides (nt),
respectively (Fig. 1a). The fragments of 39 and 20 nucleotides were purified by denaturing gel electrophoresis. We
confirmed the identity of the two fragments by demonstrating that
neither was susceptible to further RNase T1 digestion (data not
shown).
EMSA, Gel Elution, and Denaturing PAGE--
S100 macrophage
extracts and EMSA were carried out exactly as described previously
(10), except that migration was performed at 220 V for 150 min. Unless
specified, 10 fmol of labeled probe (approximately 30,000 cpm) was
added to each reaction. To elute RNA, bands were excised from the wet
gel and placed into oligonucleotide elution buffer (2 M
ammonium acetate, 1% SDS, 25 µg/ml tRNA) for 2 h at 37 °C.
RNA was precipitated and, following resuspension in water, was analyzed
on 6 M urea, 20% acrylamide gels. To elute UV cross-linked
RNA-protein complexes, dried gel slices were rehydrated in 4% ammonium
bicarbonate, 1% -mercaptoethanol, 0.1% SDS and boiled for 5 min.
Samples were left overnight on a rotating wheel and then centrifuged to
remove the debris. The supernatants were concentrated using
Ultrafree-MC columns (Millipore) and then subjected to SDS-PAGE.
UV Cross-linking and SDS-PAGE-- Samples were prepared exactly as for EMSA, except that 500,000 cpm of probe and 20 µg of protein was used and 10 µg of tRNA was included in the binding reaction. Following RNase T1 digestion (60 units) and addition of heparin, samples were placed on ice at a distance of 4 cm from a UV light source (Stratalinker) for 10 min. Samples were then treated with RNase A (0.1 mg/ml) for 10 min at room temperature, before loading onto either native gels for EMSA analysis or 9% SDS-polyacrylamide gels (37.5:1 cross-linking ratio).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Both Inducible and Constitutive Complexes Bind to a 39-nt Fragment
of the TNF- ARE--
We have shown previously that in S100 extracts
of unstimulated RAW 264.7 murine macrophages, a single protein complex
(complex 1) binding a 70-nucleotide ARE in the 3'-UTR of the TNF-
mRNA can be detected by EMSA. In similar experiments using extracts prepared from LPS-stimulated macrophages, an additional complex (complex 2) was observed that runs with a higher mobility than complex
1 (10). To further delineate the exact sequence requirements for
complex 1 and 2 formation, we digested the ARE probe with T1
ribonuclease, yielding three major fragments of 39, 20, and 10 nucleotides (Fig. 1a). The
purified 39- and 20-nt T1 products were used in an EMSA with S100
macrophage extracts as described previously (10). The 39-nucleotide
fragment supports the formation of the same complexes as those observed
previously using the complete TNF-
ARE (Fig. 1b). Binding
to the 20-nt fragment is also detectable but at a much reduced level.
These results show that both complexes 1 and 2 can form with a RNA
fragment containing clustered AUUUA pentamers. However, neither complex
forms strongly with a RNA fragment containing a single AUUUA
pentamer.
|
Identification of the RNA Sequence Involved in Formation of
Complexes 1 and 2--
To gain information on the RNA species present
in complexes 1 and 2, we performed a binding reaction with the TNF-
ARE probe and protein S100 extract. The samples were subsequently
digested with RNase T1 and loaded on a non-denaturing gel. After
migration, the RNA-protein complexes were eluted from the gel and the
purified RNA fragments were analyzed by denaturing polyacrylamide gel
electrophoresis (Fig. 2). Complex 1 mostly bound to the 39-nt fragment (see Fig. 1a). Complex 2, however, bound a number of fragments, the strongest band corresponding
to a 10-nt digestion product. This could not be further digested with
RNase T1 following elution and purification, identifying it as the
UAUUUAUUUG fragment found in the 3' end of the TNF-
ARE.
Surprisingly, only a small amount of the 39-nt fragment was eluted.
Presumably, in the conditions used for the EMSA experiment, complex 1 has a higher affinity for this sequence or is more abundant and
efficiently competes for binding. In addition, the 20-nt fragment was
clearly present, together with a 24-nt fragment that is probably a
partial digestion product containing this 20-mer sequence. These
results are consistent with the data shown in Fig. 1b, with
complex 2 having a low affinity for the 20-nt sequence, and suggest
that complex 2 is able to bind a RNA sequence containing the
pentanucleotide AUUUA flanked by several U residues.
|
Complex 2 Binds RNA Fragments Containing a UUAUUUAUU
Motif--
PCR primers were designed to generate DNA fragments with
deletions at either the 5' or 3' end of the TNF- ARE (Fig.
3a). These constructs were
subcloned and used as template for generation of labeled riboprobes.
These probes were used in EMSA analysis. Probe 3'-d1, which has the 3'
end of the TNF-
ARE deleted, supports formation of both complexes 1 and 2 in a similar fashion to the complete TNF-
ARE probe (Fig.
3b). A probe with a deletion of a further eleven nucleotides
(3'-d2) binds to complex 2, but binds very weakly to complex 1. Deletion of another 10 nucleotides (3'-d3) resulted in a probe that did
not bind, or bound weakly, to complexes 1 or 2. A construct bearing a
deletion of the 5' end of the TNF-
ARE (5'-d1) also bound to an
LPS-inducible complex, but no complex was detectable with extracts from
unstimulated cells. The mobility of this inducible complex was
consistently lower than that of complex 2 formed with the TNF-
ARE
probe. However, when unlabeled 5'-d1 RNA was used as a competitor with
labeled TNF-
ARE probe, complex 2, but not complex 1, was
specifically competed (Fig. 3c). Moreover, UV cross-linking
experiments confirmed that the complex formed with the 5'-d1 probe is
highly related to complex 2 (see below and Fig. 5). These results
indicate that complex 1 binding requires larger RNA fragments with
multiple AUUUA motifs. Complex 2 forms with smaller fragments, but its
inability to complex strongly with 3'-d3 suggests that a single AUUUA
pentamer is not the optimal binding motif.
|
|
A 55-kDa Protein Is a Component of Complex 2--
We have used the
specificity of the 5'-d1 probe for complex 2 as a means of determining
the molecular size of its protein constituents. S100 extract from
either unstimulated or stimulated macrophages was incubated with 5'-d1
probe labeled to a high specific activity. Binding was carried out in
the presence of tRNA to reduce nonspecific binding (data not shown).
Following T1 digestion and addition of heparin to reduce nonspecific
electrostatic interactions, the samples were exposed to short
wavelength UV irradiation. Proteins were separated by SDS-PAGE, and the
dried gels were autoradiographed to visualize proteins that had become
cross-linked to the radiolabeled 5'-d1 probe. A single band of
approximately 55 kDa was observed with stimulated but not with
unstimulated extracts (Fig. 5, left panel). This 55-kDa band was not detected in the absence of UV irradiation (data not shown). The same experiments were carried out
using a radiolabeled TNF- ARE probe. Again, an inducible protein of
approximately 55 kDa was observed. In addition, proteins of 48 kDa and
greater than 200 kDa cross-linked to the TNF-
ARE in a constitutive
manner.
|
The 55-kDa Protein of Complex 2 Binds to the GM-CSF ARE, but Not to the c-myc ARE-- From the results shown in Fig. 3, we predicted that complex 2 should form on the GM-CSF ARE, which contains several nonamer motifs, but not on the c-myc ARE, which contains only AUUUA pentameric motifs (Fig. 6a). To test this hypothesis, we used riboprobes containing the AREs of the GM-CSF and c-myc mRNAs in EMSA experiments. It can be seen in Fig. 6b that the GM-CSF probe supports formation of an inducible complex. No complex formation was observed using the c-myc riboprobe. In addition, a 55-kDa protein could be cross-linked to the GM-CSF probe, but only in LPS-induced extracts (Fig. 6c).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
TNF- mRNA translational regulation is governed by the
AU-rich sequence present in its 3'-UTR. In a previous report, we showed by gel retardation experiments that this AU-rich sequence is able to
form two different complexes (1 and 2) with proteins present in the
cytosolic fraction of macrophage cell extracts. Complex 1 is
characterized by a low electrophoretic mobility, is constitutively formed, is present in both nuclear and cytosolic fractions, and probably corresponds to previously reported complex A (11) and AU-A
(13). Complex 2 has a higher electrophoretic mobility and is only
detected with cytosolic extracts obtained from cells stimulated to
produce TNF-
. In this study, we characterized the sequence requirements for the formation of these two complexes.
Consistent with data concerning the specificity of complex A
(11), we showed that complex 1 is able to form with the 39-nt fragment
generated by RNase T1 digestion of the TNF- ARE probe but not with a
20-nt fragment (Figs. 1 and 2). Interestingly, the 39-nt fragment
contains five overlapping AUUUA pentamer motifs, whereas only one
pentamer is present in the 20-nt fragment. A progressive deletion of
the TNF-
ARE within the 39-mer region rapidly abolishes the
formation of complex 1 (Fig. 3b). Furthermore, complex 1 could not bind to a synthetic UUAUUUAUU sequence. Altogether, these
results suggest that complex 1 formation requires a large fragment of
the TNF-
ARE containing clustered AUUUA pentamers. The functional
significance of complex 1 remains to be elucidated. TNF-
mRNA is
both unstable and translationally repressed in quiescent macrophages
(14). As complex 1 is constitutively formed with cytosolic macrophage
extract, this complex could mediate one of these effects. AU-rich
regions, but not single AUUUA pentamers, are associated with mRNA
instability (12). Hence, there is a correlation between the sequence
requirements for mRNA degradation and those required for complex 1 formation. However, Han and co-workers (4) demonstrated that a UUAUUUAU
sequence alone is a poor mediator of translational repression. This is
consistent with the translational repression being mediated by complex
1. Further experiments will be required to elucidate the exact
relationship between complex 1, translational repression, and mRNA
destabilization.
Several lines of evidence point to the target RNA sequence involved in
the formation of the LPS-inducible complex 2 being composed of a single
(U)UAUUUAU(U) motif, but not an AUUUA pentanucleotide. This is, to our
knowledge, the first demonstration that this nonamer is by itself a
recognizable sequence motif. First, the purification of the RNA product
present in complex 2 in a gel shift experiment leads to the isolation
of a major product identified as a UAUUUAUUUG. Second, gel shift
experiments using different probes derived from the TNF- ARE
demonstrate that RNA sequences containing the UUAUUUAUU motif can form
an LPS-inducible complex (probes 3'-d2 and 5'-d1; Fig. 3). Complex 2 formation is poorly supported by a probe containing only a UAUUUAU
motif (probe 3'-d3). Third, competition experiments between an RNA
nonanucleotide and the complete TNF-
ARE show that a UUAUUUAUU motif
is more efficient than a CUAUUUAUC motif at competing for the formation
of the complex 2, whereas the CCAUUUACC nonamer does not compete at the
concentrations used (Fig. 4).
We have shown that the class II GM-CSF ARE also binds complex 2. Most Class II AREs derived from cytokine mRNA 3'-UTRs contain at least one copy of the nonanucleotide UUAUUUAUU (8). Class I AREs, which are found in several oncogene mRNAs, do not contain nonamer motifs. It is therefore unlikely that these could support complex 2 formation. Indeed, we have failed to observe an LPS-inducible complex forming on the c-myc ARE in gel retardation experiments (Fig. 6b).
From our data, it is clear that the presence of the nonamer UUAUUUAUU
motif could be a key factor in the differential recognition of class I
and II AREs by some proteins. The stabilities of class I and II
ARE-containing mRNAs are differentially regulated (8, 15). A
mutational analysis has suggested that the key feature of class II AREs
that leads to this physiological difference is reiterated AUUUA motifs,
and not UUAUUUAUU sequences. As our data show that complex 2 forms
readily on this nonamer sequence, it suggests that complex 2 is not
involved in the regulation of TNF- mRNA stability. Other class
II-specific binding activities may be responsible for this differential
regulation of mRNA stability (13). Since formation of complex 2 tightly correlates with translational derepression of the TNF-
mRNA, the nonamer motif may be a key element for translational
control. It may be that the exact physiological function of class II
AREs arises from a complex interplay between the number and positioning
of reiterated pentamers and individual nonamer motifs.
UV cross-linking experiments using the 5'-d1 probe specific for complex 2 clearly demonstrate that this complex contains a 55-kDa protein. Using the same technical approach, several groups have described proteins that bind to the AU-rich element of cytokine mRNAs in different cellular systems. With the exception of the work of Bohjanen et al., all these data concerned constitutive ARE binding activities. Bohjanen et al. (13) described 30-kDa (AU-B) and 43-kDa (AU-C) class II ARE-specific binding activities that were only present following activation of T cells. Unlike complex 2, these activities co-migrated with a constitutive ARE binding activity in gel retardation experiments. On these bases, we believe that complex 2 contains a previously undescribed inducible 55-kDa ARE-binding protein.
This work forms the basis of an analysis into the relationship between
translational control and mRNA stability in macrophages, and
demonstrates further differences between cytokine and oncogene AREs.
Isolation of the 55-kDa protein by using the specific RNA probes
described in this report should provide further information as to its
role in the translational control of TNF- mRNA.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Chantal Bourget for technical assistance, Dr. C. Cazenave for helpful discussion, and Dr. L. Droogmans for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by EC Biotech Program Grant BIO4-CT95-0045.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.
¶ Supported by a Televie grant.
To whom correspondence should be addressed. Tel.: 322-6509816;
Fax: 322-6509839; E-mail: vkruys{at}dbm.ulb.ac.be.
1
The abbreviations used are: UTR, untranslated
region; ARE, AU-rich element; TNF-, tumor necrosis factor-
; LPS,
lipopolysaccharide; GM-CSF, granulocyte/macrophage colony-stimulating
factor; PAGE, polyacrylamide gel electrophoresis; EMSA, electrophoretic
mobility shift assay; nt, nucleotide(s).
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|