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Gayatri Gowrishankar,1, Frank Bollig,2,
Natalie Redich,1 Klaus Resch,2 and Helmut Holtmann1*
Institute of Biochemistry,1 Institute of Pharmacology, Medical School Hannover, D-30625 Hannover, Germany2
Received 21 November 2003/ Returned for modification 18 December 2003/ Accepted 12 March 2004
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ARE-dependent rapid decay is the target of several different signaling mechanisms. These include the Jun N-terminal kinase (7, 27), phosphatidylinositol-3-kinase (28), and p38 mitogen-activated protein (MAP) kinase pathways (12, 29, 34, 46). Activation of p38 MAP kinase, triggered by the inflammatory cytokine interleukin-1 (IL-1) or by bacterial lipopolysaccharide, induces stabilization of ARE-containing endogenous mRNAs and reporter mRNAs containing several different AREs, including those of IL-3, IL-6, IL-8, tumor necrosis factor alpha (TNF-
), cyclooxygenase 2, and c-fos. Experiments with cultured cells and with mice point to involvement of the p38 MAP kinase substrate MAP kinase-activated protein kinase 2 (MK2) in this effect of the p38 MAP kinase pathway (19, 22, 31, 46). Results obtained so far indicate that stabilization induced by this pathway is selective for certain AU-rich transcripts, whereas no stabilization is observed with certain other ARE-containing mRNAs (16, 22), as well as with short-lived mRNAs lacking AREs (4). The exact requirements for p38/MK2-induced stabilization in terms of both cis-acting sequences and trans-acting factors have not yet been defined.
mRNA decay begins with loss of the poly(A) tail (deadenylation), and the AREs were observed to enhance deadenylation (11, 38, 44). More recently, AREs have been suggested to mediate the recruitment of the exosome, a complex of RNases involved in 3'-to-5' degradation of the mRNA body (8, 30). A number of proteins have been identified that can interact with AREs and/or affect the stability of ARE-containing transcripts. The most prominent among them include HuR, TTP, BRF1, KSRP, AUF1/hnRNP D, and other members of the hnRNP family of proteins. Except for HuR, all of the above-mentioned proteins can function as destabilizers of the ARE mRNA to which they bind. Since activation of the p38/MK2 pathway selectively stabilizes ARE mRNAs, the ARE binding proteins are likely targets of the pathway.
We previously observed in gel shift assays that HuR interacts with 3' UTR sequences of IL-8 (4), a cytokine that is chemotactic for leukocytes, activates neutrophils and is involved in angiogenesis. HuR is a ubiquitously expressed member of the human ELAV family of RNA binding proteins (24) whose functional role in the stabilization of several ARE-containing mRNAs has been established (15, 23, 33, 35, 42, 43). It also plays a role in the nuclear export of mRNA (17) and in control of translation (20, 26).
Here we report that the IL-8 3' UTR contains an ARE with two domains that differ in function. Destabilization by this ARE and mutant forms thereof is counteracted by p38/MK2 activation but not by HuR overexpression. Instead, HuR-induced stabilization can be imposed by sequences not required for responsiveness to p38/MK2. The results demonstrate that AREs can consist of different domains with different functions in destabilization and stabilization induced by different mechanisms.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, in which nt 1017 to 1076 are deleted, the PCR-generated nt 1077-to-1310 fragment with a 5' NheI site was inserted into the BglII site of ptet-BBB and then the nt 972-to-1016 fragment was inserted into the NheI site. cDNAs corresponding to the AREs of IL-8 and GM-CSF (accession no. M10663), as well as parts thereof, were also cloned into the BglII site of pBluescript for in vitro transcription (see below). Mutations of AUUUA motifs were performed by exchanging the central U for G by using the QuikChange kit (Stratagene). To express ß-globin transcripts without a poly(A) tail, the polyadenylation signal was mutated to generate an AvaIII site and an RT-PCR-amplified, AvaIII-flanked DNA of the stem-loop of the human histone 1.3 gene and 3' flanking sequences were inserted (ptet-BBB-HSL). ptet-BBB-IL-8ARE-HSL was generated by inserting nt 1017 to 1076 of IL-8 as described above. Expression plasmids for myc-tagged HuR and constitutively active MAP kinase kinase 6 (MKK62E) have been described previously (10, 46). Transfection and determination of mRNA decay. HeLa cells constitutively expressing the tetracycline-controlled transactivator protein (18; kindly provided by H. Bujard) were cultured in Dulbecco's modified Eagle medium complemented with 5% fetal calf serum. Transient transfections by the calcium phosphate method and RNA degradation kinetics were performed as previously described (46). Briefly, cells (4 x 106 seeded per 9-cm-diameter dish) were transfected with the indicated plasmids. Amounts of plasmid DNA within each experiment were kept constant by adding empty vector. For kinetics of mRNA degradation, cells from one dish were trypsinized following transfection and distributed into parallel cultures. The next day, transcription from the tetracycline-regulatable promoter was stopped by addition of doxycycline (3 µg/ml). At the indicated times thereafter, total RNA was isolated and Northern blot analysis was performed with digoxigenin-labeled antisense RNA probes. RNA half-lives were determined as described in reference 46, with a video imaging system and the Molecular Analyst program (Bio-Rad).
Analysis of poly(A) tail length. RNA samples were mixed with an equal amount of formamide loading buffer (80% [vol/vol] formamide and 1 mg of bromophenol blue per ml in 1x TBE [89 mM Tris-borate, 2 mM EDTA, pH 8.3]), incubated at 65°C for 15 min, and loaded onto denaturing polyacrylamide gels (4% [wt/vol] acrylamide, 7 M urea). Running in 1x TBE was performed at about 10 V/cm for various times, depending on the size of the RNA monitored. Semidry electroblotting (Peqlabs) at 200 mA for 1 h with 1x TBE as buffer was used to transfer the RNAs to a nylon membrane for Northern blot analysis. Deadenylated transcripts were prepared in vitro by mixing total RNA from transfected cells with oligo(dT) (0.5 µg for every 10 µg of total RNA) in buffer containing 200 mM KCl and 1 mM EDTA (pH 8.0). The sample was incubated at 90°C for 2 min and then annealed at 25°C for 10 min. Two volumes of a solution containing 100 mM KCl, 0.5 mM EDTA (pH 8.0), 20 mM Tris-HCl (pH 8.0), 28 mM MgCl2, 20 U of RNase inhibitor, and 1 U of RNase H were then added. The sample was incubated at 37°C for 30 min and precipitated with ethanol.
Preparation of cytoplasmic extracts. All steps were carried out in the cold. HeLa cells (106 per sample) were washed once with phosphate-buffered saline, harvested by scraping, pelleted by centrifugation, and resuspended in 200 µl of hypotonic buffer (10 mM HEPES [pH 7.9], 10 mM KCl, 1.5 mM MgCl2, 1 µg of leupeptin per ml, 1 µg of aprotinin per ml, 0.5 mM phenylmethylsulfonyl fluoride). A 25-µl volume of the same buffer including 2.5% (vol/vol) Nonidet P-40 was then added. After centrifugation at 1,000 x g for 4 min, the supernatants were removed and cleared by centrifugation at 10,000 x g. Aliquots were frozen at –70°C.
In vitro transcription and electrophoretic mobility shift assays.
Labeled RNA (107 to 108 cpm/µg) was synthesized by incubating 1 to 3 µg of linearized plasmid DNA in a mixture of 50 µCi of [-32P]UTP (400 Ci/mmol; Hartmann Analytic); 18 µM unlabeled UTP; 0.5 mM unlabeled ATP, CTP, and GTP; 2 U of T7 or T3 RNA polymerase per µl, as required (both enzymes were from Roche); and 2 U of RNase inhibitor (MBI) per µl for 1 h at 37°C. RNase-free DNase (Roche) was then added to a final concentration of 1 U/µl. After incubation for 15 min at 37°C, the RNA was passed through a NucTrap push column (Stratagene) to remove free nucleotides and stored at –80°C. Radiolabeled RNA probes (1.5 x 105 cpm) were incubated with cytoplasmic extracts (6 µg of protein per sample) in 20 µl of buffer containing 20 mM HEPES (pH 7.9), 100 mM KCl, 2 mM MgCl2, 3% (vol/vol) glycerol, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 5 µg of pepstatin A per ml, and 200 µg of tRNA per ml for 10 min at 30°C. RNase T1 (30 U/sample) was then added, and incubation was continued for 20 min at 37°C. Where indicated, mouse monoclonal antibody 19F12 against HuR (0.75 µg, kindly donated by Henry Furneaux) or rabbit antiserum against hnRNP D/AUF1 (diluted 1:200; a gift of Gary Brewer) was included for the last 10 min. Samples were electrophoresed on a nondenaturing polyacrylamide gel (5% acrylamide in 0.25x TBE buffer). The gels were dried and autoradiographed.
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RESULTS |
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Further investigation showed that these two domains differ in function. An RNA that contained only the auxiliary domain (Fig. 2A, nt 972 to 1048) was stable, indicating that this domain cannot destabilize on its own. Lack of destabilization was also observed upon insertion of a fragment that contains only the AUUUA-containing core domain and lacks the auxiliary domain (Fig. 4C, construct IL-8 CD). However, owing to the site of insertion in the ß-globin construct, the fragments are located directly after the stop codon. To account for a possible effect of close proximity to the stop codon, the half-life of the construct containing just the core domain was compared to that of a construct in which spacing from the stop codon was preserved by inserting a random sequence identical in length to the 5' domain. That construct (IL-8 R-CD)—in contrast to the one without spacing—clearly exhibits destabilization (Fig. 4C). Yet destabilization is much weaker compared to that of an RNA with the complete ARE (compare IL-8 R-CD and AD-CD in Fig. 4C). Thus, spacing between the stop codon and a destabilizing AU-rich sequence might be critical for its functioning. Lack of destabilization by the auxiliary domain alone is not related to that, since in the nt 972-to-1048 construct (Fig. 2A) there is a distance of about 50 nt between the auxiliary domain and the stop codon. Taken together, the results show that the 5' part of the IL-8 ARE functions as an auxiliary domain that does not destabilize on its own but strongly enhances the moderate destabilization exerted by the core domain. Replacement of 10 nt in the 5', central, or 3' part of the auxiliary domain with a 10-nt random sequence resulted in loss of destabilizing activity, with the strongest impairment noted when the central part was replaced (Fig. 4D). This indicates that sequences most crucial for enhancing destabilization are located in the central part of the auxiliary domain.
Importantly, activation of the p38 MAP kinase cascade by MKK62E induced further stabilization of the RNA containing the random sequence plus the core domain (IL-8 R-CD, Fig. 4C). Thus, the AUUUA motif-containing core domain is sufficient to confer sensitivity to p38 MAP kinase-induced stabilization.
Evidence for a bipartite structure has been presented for the GM-CSF ARE (47) (Fig. 4A), which mediates extents of destabilization and MKK62E-induced stabilization very similarly to the IL-8 ARE (46; Fig. 4C). As observed for the IL-8 ARE, replacement of the auxiliary domain with a random sequence resulted in diminished destabilization. Furthermore, this mRNA was also stabilized by coexpression of MKK62E, confirming the role of the AUUUA-containing domain in p38/MK2-induced stabilization (Fig. 4C).
Role of AUUUA motifs in destabilization and p38 MAP kinase-induced stabilization.
While the GM-CSF ARE contains five overlapping AUUUA motifs, the IL-8 ARE contains four, with only two of them overlapping. It contains no UUAUUUA(U/A)(U/A) nonamer, which has been described as a key destabilizing sequence (21, 50). To investigate the contributions of the individual AUUUA motifs, they were destroyed singly and in combinations by introducing U
G point mutations of the central U (Fig. 5A contains a scheme of the mutant sequences analyzed). Elimination of the first motif resulted in significantly decreased destabilizing efficiency of the ARE, compared to that of the intact ARE without (data not shown) or with (Fig. 5B, compare transcript M1 to IL-8972-1310) adjacent sequences. Destroying each of the other motifs separately (Fig. 5B, control for mutants M2 to M4) also induced marked loss of destabilization. Progressively mutating the remaining AUUUA motifs in the complete ARE, yielding RNAs with two intact motifs (data not shown), only one intact motif (mutants M123 and M234), or no intact motif left (M1234) resulted in progressive loss of the destabilizing effect. Unexpectedly, destroying the motif overlap by mutating motif 3 or 4 or both did not result in a more pronounced loss of destabilizing activity. Thus, no evidence was obtained for a superior function of overlapping as opposed to dispersed motifs. Rather, Fig. 5 and additional experiments show a tendency to more strongly compromised function owing to loss of motif 1. Importantly, in all of the mutants, MKK62E expression still induced stabilization of the RNA (Fig. 5B). It is noteworthy that while the half-life of the rapidly degraded reporters containing the AREs of GM-CSF and IL-8 is extended to about 2 to 3 h (Fig. 4C), the half-life of the IL-8 ARE transcripts containing single mutations in their AUUUA motifs, which is about 2 h in unstimulated cells, is extended to more than 6 h (Fig. 5). Even for mutant M1234—which has only very weak residual destabilizing capacity—stabilization in response to activation of the p38/MK2 pathway was clearly discernible (half-life of 8 h versus 4.2 h without MKK62E). This demonstrates that RNA elements without an intact AUUUA motif can mediate p38 MAP kinase-regulated mRNA stability.
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Interaction of the ARE with cytoplasmic proteins involves both domains and is not affected by p38 activation. Gel shift assays with radiolabeled RNA representing the complete IL-8 ARE show two prominent and additional minor complexes formed between the RNA and HeLa cytoplasmic proteins (Fig. 6A). Mutating one AUUUA motif (mutant M3) resulted in a slight loss of the intensity of the complexes, whereas mutating all four motifs (M1234) caused a dramatic loss of the major complexes. Thus, intensity of complex formation correlates with the extent of destabilization. No change in complex formation was observed when activating the p38/MK2 pathway by MKK62E (Fig. 6A). Complex formation was impaired, resembling the result obtained with the M1234 mutant, when assaying RNA corresponding to the auxiliary domain alone or the core domain alone (Fig. 6B). Thus, both domains contribute to formation of the two major protein complexes. Furthermore, comparing the complete GM-CSF ARE and its two domains separately also shows a different and weaker interaction with proteins for both domains. To identify the proteins in the complexes, antibodies against the ARE-binding proteins hnRNP D/AUF1 and HuR were included in the assay (Fig. 6B). Anti-hnRNP D/AUF1 antibodies did not affect complex formation with the IL-8 ARE, whereas they induced a shift with the GM-CSF ARE, serving as a positive control. Antibodies against HuR induced a supershift with both AREs (Fig. 6B), indicating that they both can interact with HuR.
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DISCUSSION |
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Results in Fig. 4 show a two-domain structure of the 60-nt region. A core domain containing four AUUUA motifs apparently plays an important role in the rapid decay of the IL-8 mRNA, since a truncated transcript that lacks that domain has a much longer half-life (about 2 h compared to <20 min, Fig. 1). It is possible that the 2-h half-life reflects the function of additional destabilizing elements, as has been observed for the TNF- mRNA (39). Importantly, stabilization in response to p38/MK2 signaling is completely lost in the mRNA lacking the core domain. This indicates that the core domain is essential for regulation by this pathway.
Our own previous studies (46) and a report on the effect of 3' UTR regions of IL-8 on chloramphenicol acetyltransferase expression (49) indicated, however, that the AUUUA motif-containing core region alone has low destabilizing activity. We now identify a 30-nt region immediately 5' to the core as a potent codestabilizing domain. A two-domain structure has been reported for the ARE of c-fos (6) that consists of a 5' domain containing three AUUUA motifs and a U-rich 3' domain. The IL-8 ARE is organized more similarly to the GM-CSF and IL-3 AREs, which contain clusters of overlapping AUUUA motifs in the 3' part and AU-rich sequences 5' to the AUUUA motifs that contribute to destabilization (47). Evidence for the importance of upstream sequences has also been presented for the TNF- ARE (40). Interestingly—unlike the upstream region of the IL-8 ARE—that of the GM-CSF ARE contains two AUUUA motifs which, however, are dispensable for its destabilizing activity (47). Both upstream domains are A and U rich (100% for GM-CSF, 81% for IL-8). Further characteristics of auxiliary domains have to be defined in future studies. The importance of such domains could be to provide buffer capacity against mutations of AUUUA motifs (Fig. 5B and reference 6).
While the auxiliary domains of IL-8 and GM-CSF strongly contribute to destabilization, they are dispensable for the stabilizing effect of the p38/MK2 pathway. Furthermore, severely compromising ARE function by mutating the AUUUA motifs—which results in reduced interaction with proteins (Fig. 6A), presumably including those involved in destabilization—did not yield RNAs that lose responsiveness to p38/MK2. A database for ARE-containing transcripts has been established on the basis of the sequence WWWUAUUUAUWWW and allowing one mismatch on either side of the UAUUUAU motif (2). In the IL-8 ARE, the second and third AUUUA motifs form the centers of sequences fulfilling these criteria. Interestingly, mutants in which both of these AUUUA motifs are destroyed still mediate destabilization and MKK6-induced stabilization. Thus, the sequence requirements for this type of regulation are even more flexible than initially assumed and can be met by transcripts not included in the ARED database. The results presented in Fig. 4 and 5 show that an optimal ARE function requires the context of several AUUUA motifs plus an upstream domain. Mutating any of these components results in impaired destabilization. Importantly, in all cases in which significant destabilization occurred, it was inhibited by activation of p38/MK2. This was even true for ARE sequences without an AUUUA motif. In unstimulated cells, the respective mRNA, containing the M1234 mutant ARE, was only moderately destabilized compared to the ß-globin mRNA with no insertion and thus lost the physiologically important feature of allowing a sharp peak of gene expression in response to stimulation. However, in other mRNAs, ARE-like elements may exist in which the lack of AUUUA motifs is compensated for by even more effective auxiliary domains.
The overall picture that emerges from these results is that as long as there is measurable destabilization through an ARE, activation of p38/MK2 induces stabilization. On the other hand, short-lived mRNAs that lack AREs are not affected by p38/MK2 activation (4). These observations are compatible with a model in which this signaling pathway—rather than activating an independent stabilizing mechanism—impairs a distinct mechanism of destabilization that is dependent on the recognition of an ARE by a certain protein(s).
A likely possibility is that MK2 phosphorylates and thereby compromises the function of a protein directly involved in destabilization. Our observation that expressing MKK62E does not affect RNA-protein complex formation (Fig. 6A) suggests a functional change other than binding to the ARE. Candidate proteins for which evidence has been presented that they are phosphorylated by MK2 and selectively interact with AREs include hnRNP A0 (36), PABP1 (3), and TTP (25).
It has to be noted, however, that not all AREs mediate stabilization through this pathway. A number of mRNAs fulfilling the criteria of the ARED database are not stabilized in a p38 MAP kinase-dependent manner in response to lipopolysaccharide as an activator of p38/MK2 signaling (16). There could be several not mutually exclusive reasons for this observation, one of them being that certain AREs lack features that we have not yet identified. Another possible reason is that additional instability determinants not responsive to p38/MK2-induced stabilization override the effect of that signaling mechanism. This appears quite likely in view of our results showing rather flexible requirements for this type of stabilization, and indeed such a situation has been shown recently for the TNF- mRNA (39), which contains a constitutive decay element. Such a mechanism can impose selectivity on stabilization within a broad range of transcripts whose AREs may fulfill the requirements for p38/MK2-induced stabilization.
In this paper, we demonstrate a fundamental difference between sequence requirements for stabilization through the p38/MK2 pathway as opposed to stabilization due to increased cytoplasmic HuR. HuR has been found to interact with several AREs in vitro and in cells, and its overexpression induced stabilization of mRNAs for vascular endothelial growth factor (23), nitric oxide synthase II (35), eotaxin (1), and different ARE-containing reporter RNAs (14, 15, 33). Furthermore, suppression of HuR amounts by antisense RNA or small interfering RNA approaches inhibited stabilization induced by hypoxia (23), cytokines (35), or UV light (43) or during cell cycle progression (42). Most recently, constitutively active MK2 (41), as well as stimuli known to activate the p38/MK2 pathway (H2O2 [41] and TNF- [1]), was found to induce cytoplasmic accumulation of HuR. Thus, HuR could play a role in p38/MK2-induced stabilization. The lack of stabilization of an IL-8 ARE reporter RNA (Fig. 7), however, argues against this possibility. Furthermore, an AUUUA motif-containing core domain clearly allows stabilization of the mRNA by the p38/MK2 pathway, as shown for the core domains of GM-CSF and IL-8 (Fig. 4), whereas it does not suffice to allow stabilization by HuR. Rather, a separate domain can impose stabilization through HuR, as shown for the c-fosAD-IL-8CD chimeric ARE (Fig. 7). Different mechanisms of stabilization are also supported by the characteristics of the stabilized transcripts. HuR has been shown to prevent decay of the RNA body, rather than deadenylation (33).
The observation that HuR binds to the IL-8 ARE in vitro yet does not stabilize an IL-8 ARE-containing reporter RNA when overexpressed can be explained in different ways. For example, the affinity of HuR for the IL-8 ARE may be too low, or HuR may have to bind in a distinct way and/or in combination with certain other proteins to stabilize an RNA. Alternatively, binding in cells might be prevented by proteins that do not bind to the GM-CSF ARE or the c-fos ARE. Irrespective of the reason, the absence of a stabilizing effect of HuR on IL-8 ARE-containing mRNA argues against increased levels of HuR in the cytoplasm as the sole mechanistic basis for p38/MK2-induced stabilization. In additional experiments (data not shown), activation of p38/MK2 in cells overexpressing HuR did not result in increased stabilization of an IL-8 ARE reporter RNA compared to that in cells without ectopically expressed HuR. Taken together, these results suggest that HuR and p38/MK2 activation can induce mRNA stabilization by two independent mechanisms. This does not exclude the possibility that—as an additional mode of stabilization—p38/MK2 activation might also stabilize indirectly through HuR by inducing its cytoplasmic translocation (41).
In summary, our data show that AREs can consist of functionally distinct domains (Fig. 8). The IL-8 and GM-CSF AREs contain a core domain with several AUUUA motifs and moderate destabilizing activity by a mechanism sensitive to inhibition by p38/MK2 activation. The 5' auxiliary domain strongly enhances the moderate destabilizing activity of the core domain and thus is mandatory for most rapid mRNA degradation, the hallmark of ARE-mediated regulation. Stabilization by HuR requires features absent in the IL-8 ARE but present in auxiliary domains of GM-CSF and c-fos. The fact that mRNA stabilization by the p38/MK2 kinase cascade and that by HuR require differing sequence characteristics and consequently have different transcript selectivities suggests that the two modes of stabilization can operate in different physiological situations.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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R.W. and G.G. contributed equally to this work.
Present address: Biocenter, University of Wuerzburg, Physiological Chemistry I, D-97074 Wuerzburg, Germany.
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, control; 
, MKK62E for IL-8 R-CD). (D) Destabilizing activity of the IL-8 ARE (IL-8AD-CD) and derivatives in which the auxiliary domain was mutated by exchanging 10 nt of its 5' part (IL-8AD(R1-10)-CD), its central part (IL-8AD(R11-20)-CD), or its 3' part (IL-8AD(R21-30)-CD) with a random sequence (GCCGCCUCGA). mRNA stability was assayed and quantified as described in the legend to Fig. 
