Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 2004 Feb 1;377(Pt 3):629–639. doi: 10.1042/BJ20031484

Structural and functional dissection of a conserved destabilizing element of cyclo-oxygenase-2 mRNA: evidence against the involvement of AUF-1 [AU-rich element/poly(U)-binding/degradation factor-1], AUF-2, tristetraprolin, HuR (Hu antigen R) or FBP1 (far-upstream-sequence-element-binding protein 1).

Gareth Sully 1, Jonathan L E Dean 1, Robin Wait 1, Lesley Rawlinson 1, Tomas Santalucia 1, Jeremy Saklatvala 1, Andrew R Clark 1
PMCID: PMC1223914  PMID: 14594446

Abstract

COX-2 (cyclo-oxygenase-2) mRNA is degraded rapidly in resting cells, but is stabilized by the mitogen-activated protein kinase p38 signalling pathway in response to pro-inflammatory stimuli. A conserved ARE (AU-rich element) of the COX-2 3' untranslated region, CR1 (conserved region 1), acts as a potent instability determinant, and mediates stabilization in response to p38 activation. A detailed structural and functional analysis of this element was performed in an attempt to identify RNA-binding proteins involved in the regulation of COX-2 mRNA stability. Destabilization of a beta-globin reporter mRNA was dependent upon two distinct AREs within CR1, each containing three copies of the sequence AUUUA. CR1 was shown to bind AUF-1 [ARE/poly(U)-binding/degradation factor-1] and/or AUF-2, HuR (Hu antigen R), TTP (tristetraprolin) and FBP1 (far-upstream-sequence-element-binding protein 1), yet these factors did not appear to account for the effects of CR1 upon mRNA stability. Mutant sequences were identified that were incapable of destabilizing a reporter mRNA, yet showed unimpaired binding of FBP1 and AUF-1 and/or -2. TTP was absent from the HeLa cell line used in this analysis. Finally, RNA interference experiments argued against a prominent role for HuR in the CR1-mediated regulation of mRNA stability. We conclude that at least one critical regulator of COX-2 mRNA stability is likely to remain unidentified at present.

Full Text

The Full Text of this article is available as a PDF (278.8 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bachelor Michael A., Silvers Amy L., Bowden Gim T. The role of p38 in UVA-induced cyclooxygenase-2 expression in the human keratinocyte cell line, HaCaT. Oncogene. 2002 Oct 10;21(46):7092–7099. doi: 10.1038/sj.onc.1205855. [DOI] [PubMed] [Google Scholar]
  2. Blaxall Burns C., Pende Aldo, Wu Steven C., Port J. David. Correlation between intrinsic mRNA stability and the affinity of AUF1 (hnRNP D) and HuR for A+U-rich mRNAs. Mol Cell Biochem. 2002 Mar;232(1-2):1–11. doi: 10.1023/a:1014819016552. [DOI] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Brennan C. M., Steitz J. A. HuR and mRNA stability. Cell Mol Life Sci. 2001 Feb;58(2):266–277. doi: 10.1007/PL00000854. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carballo E., Cao H., Lai W. S., Kennington E. A., Campbell D., Blackshear P. J. Decreased sensitivity of tristetraprolin-deficient cells to p38 inhibitors suggests the involvement of tristetraprolin in the p38 signaling pathway. J Biol Chem. 2001 Sep 6;276(45):42580–42587. doi: 10.1074/jbc.M104953200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carballo E., Lai W. S., Blackshear P. J. Feedback inhibition of macrophage tumor necrosis factor-alpha production by tristetraprolin. Science. 1998 Aug 14;281(5379):1001–1005. doi: 10.1126/science.281.5379.1001. [DOI] [PubMed] [Google Scholar]
  7. Chen C. Y., Gherzi R., Ong S. E., Chan E. L., Raijmakers R., Pruijn G. J., Stoecklin G., Moroni C., Mann M., Karin M. AU binding proteins recruit the exosome to degrade ARE-containing mRNAs. Cell. 2001 Nov 16;107(4):451–464. doi: 10.1016/s0092-8674(01)00578-5. [DOI] [PubMed] [Google Scholar]
  8. Clark Andrew R., Dean Jonathan L. E., Saklatvala Jeremy. Post-transcriptional regulation of gene expression by mitogen-activated protein kinase p38. FEBS Lett. 2003 Jul 3;546(1):37–44. doi: 10.1016/s0014-5793(03)00439-3. [DOI] [PubMed] [Google Scholar]
  9. Cok S. J., Morrison A. R. The 3'-untranslated region of murine cyclooxygenase-2 contains multiple regulatory elements that alter message stability and translational efficiency. J Biol Chem. 2001 Apr 9;276(25):23179–23185. doi: 10.1074/jbc.M008461200. [DOI] [PubMed] [Google Scholar]
  10. Coussens Lisa M., Werb Zena. Inflammation and cancer. Nature. 2002 Dec 19;420(6917):860–867. doi: 10.1038/nature01322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Davis-Smyth T., Duncan R. C., Zheng T., Michelotti G., Levens D. The far upstream element-binding proteins comprise an ancient family of single-strand DNA-binding transactivators. J Biol Chem. 1996 Dec 6;271(49):31679–31687. doi: 10.1074/jbc.271.49.31679. [DOI] [PubMed] [Google Scholar]
  12. Dean J. L., Brook M., Clark A. R., Saklatvala J. p38 mitogen-activated protein kinase regulates cyclooxygenase-2 mRNA stability and transcription in lipopolysaccharide-treated human monocytes. J Biol Chem. 1999 Jan 1;274(1):264–269. doi: 10.1074/jbc.274.1.264. [DOI] [PubMed] [Google Scholar]
  13. Dean J. L., Wait R., Mahtani K. R., Sully G., Clark A. R., Saklatvala J. The 3' untranslated region of tumor necrosis factor alpha mRNA is a target of the mRNA-stabilizing factor HuR. Mol Cell Biol. 2001 Feb;21(3):721–730. doi: 10.1128/MCB.21.3.721-730.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dean Jonathan L. E., Sarsfield Simon J., Tsounakou Elizabeth, Saklatvala Jeremy. p38 Mitogen-activated protein kinase stabilizes mRNAs that contain cyclooxygenase-2 and tumor necrosis factor AU-rich elements by inhibiting deadenylation. J Biol Chem. 2003 Jul 25;278(41):39470–39476. doi: 10.1074/jbc.M306345200. [DOI] [PubMed] [Google Scholar]
  15. Dean Jonathan L. E., Sully Gareth, Wait Robin, Rawlinson Lesley, Clark Andrew R., Saklatvala Jeremy. Identification of a novel AU-rich-element-binding protein which is related to AUF1. Biochem J. 2002 Sep 15;366(Pt 3):709–719. doi: 10.1042/BJ20020402. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Dixon D. A., Tolley N. D., King P. H., Nabors L. B., McIntyre T. M., Zimmerman G. A., Prescott S. M. Altered expression of the mRNA stability factor HuR promotes cyclooxygenase-2 expression in colon cancer cells. J Clin Invest. 2001 Dec;108(11):1657–1665. doi: 10.1172/JCI12973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Dixon Dan A., Balch Glen C., Kedersha Nancy, Anderson Paul, Zimmerman Guy A., Beauchamp R. Daniel, Prescott Stephen M. Regulation of cyclooxygenase-2 expression by the translational silencer TIA-1. J Exp Med. 2003 Jul 28;198(3):475–481. doi: 10.1084/jem.20030616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Duncan R., Bazar L., Michelotti G., Tomonaga T., Krutzsch H., Avigan M., Levens D. A sequence-specific, single-strand binding protein activates the far upstream element of c-myc and defines a new DNA-binding motif. Genes Dev. 1994 Feb 15;8(4):465–480. doi: 10.1101/gad.8.4.465. [DOI] [PubMed] [Google Scholar]
  19. Ford L. P., Watson J., Keene J. D., Wilusz J. ELAV proteins stabilize deadenylated intermediates in a novel in vitro mRNA deadenylation/degradation system. Genes Dev. 1999 Jan 15;13(2):188–201. doi: 10.1101/gad.13.2.188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Frevel Mathias A. E., Bakheet Tala, Silva Aristobolo M., Hissong John G., Khabar Khalid S. A., Williams Bryan R. G. p38 Mitogen-activated protein kinase-dependent and -independent signaling of mRNA stability of AU-rich element-containing transcripts. Mol Cell Biol. 2003 Jan;23(2):425–436. doi: 10.1128/MCB.23.2.425-436.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gu Wei, Pan Feng, Zhang Honglai, Bassell Gary J., Singer Robert H. A predominantly nuclear protein affecting cytoplasmic localization of beta-actin mRNA in fibroblasts and neurons. J Cell Biol. 2002 Jan 7;156(1):41–51. doi: 10.1083/jcb.200105133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Guhaniyogi J., Brewer G. Regulation of mRNA stability in mammalian cells. Gene. 2001 Mar 7;265(1-2):11–23. doi: 10.1016/s0378-1119(01)00350-x. [DOI] [PubMed] [Google Scholar]
  23. He L., Weber A., Levens D. Nuclear targeting determinants of the far upstream element binding protein, a c-myc transcription factor. Nucleic Acids Res. 2000 Nov 15;28(22):4558–4565. doi: 10.1093/nar/28.22.4558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Irwin N., Baekelandt V., Goritchenko L., Benowitz L. I. Identification of two proteins that bind to a pyrimidine-rich sequence in the 3'-untranslated region of GAP-43 mRNA. Nucleic Acids Res. 1997 Mar 15;25(6):1281–1288. doi: 10.1093/nar/25.6.1281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lagnado C. A., Brown C. Y., Goodall G. J. AUUUA is not sufficient to promote poly(A) shortening and degradation of an mRNA: the functional sequence within AU-rich elements may be UUAUUUA(U/A)(U/A). Mol Cell Biol. 1994 Dec;14(12):7984–7995. doi: 10.1128/mcb.14.12.7984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lasa M., Mahtani K. R., Finch A., Brewer G., Saklatvala J., Clark A. R. Regulation of cyclooxygenase 2 mRNA stability by the mitogen-activated protein kinase p38 signaling cascade. Mol Cell Biol. 2000 Jun;20(12):4265–4274. doi: 10.1128/mcb.20.12.4265-4274.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mahtani K. R., Brook M., Dean J. L., Sully G., Saklatvala J., Clark A. R. Mitogen-activated protein kinase p38 controls the expression and posttranslational modification of tristetraprolin, a regulator of tumor necrosis factor alpha mRNA stability. Mol Cell Biol. 2001 Oct;21(19):6461–6469. doi: 10.1128/MCB.21.9.6461-6469.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Min H., Turck C. W., Nikolic J. M., Black D. L. A new regulatory protein, KSRP, mediates exon inclusion through an intronic splicing enhancer. Genes Dev. 1997 Apr 15;11(8):1023–1036. doi: 10.1101/gad.11.8.1023. [DOI] [PubMed] [Google Scholar]
  29. Mukhopadhyay Debnath, Houchen Courtney W., Kennedy Susan, Dieckgraefe Brian K., Anant Shrikant. Coupled mRNA stabilization and translational silencing of cyclooxygenase-2 by a novel RNA binding protein, CUGBP2. Mol Cell. 2003 Jan;11(1):113–126. doi: 10.1016/s1097-2765(03)00012-1. [DOI] [PubMed] [Google Scholar]
  30. Myer V. E., Fan X. C., Steitz J. A. Identification of HuR as a protein implicated in AUUUA-mediated mRNA decay. EMBO J. 1997 Apr 15;16(8):2130–2139. doi: 10.1093/emboj/16.8.2130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nabors L. B., Gillespie G. Y., Harkins L., King P. H. HuR, a RNA stability factor, is expressed in malignant brain tumors and binds to adenine- and uridine-rich elements within the 3' untranslated regions of cytokine and angiogenic factor mRNAs. Cancer Res. 2001 Mar 1;61(5):2154–2161. [PubMed] [Google Scholar]
  32. Peng S. S., Chen C. Y., Xu N., Shyu A. B. RNA stabilization by the AU-rich element binding protein, HuR, an ELAV protein. EMBO J. 1998 Jun 15;17(12):3461–3470. doi: 10.1093/emboj/17.12.3461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rehbein Monika, Wege Konstanze, Buck Friedrich, Schweizer Michaela, Richter Dietmar, Kindler Stefan. Molecular characterization of MARTA1, a protein interacting with the dendritic targeting element of MAP2 mRNAs. J Neurochem. 2002 Sep;82(5):1039–1046. doi: 10.1046/j.1471-4159.2002.01058.x. [DOI] [PubMed] [Google Scholar]
  34. Ridley S. H., Dean J. L., Sarsfield S. J., Brook M., Clark A. R., Saklatvala J. A p38 MAP kinase inhibitor regulates stability of interleukin-1-induced cyclooxygenase-2 mRNA. FEBS Lett. 1998 Nov 13;439(1-2):75–80. doi: 10.1016/s0014-5793(98)01342-8. [DOI] [PubMed] [Google Scholar]
  35. Rousseau Simon, Morrice Nick, Peggie Mark, Campbell David G., Gaestel Matthias, Cohen Philip. Inhibition of SAPK2a/p38 prevents hnRNP A0 phosphorylation by MAPKAP-K2 and its interaction with cytokine mRNAs. EMBO J. 2002 Dec 2;21(23):6505–6514. doi: 10.1093/emboj/cdf639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sawaoka Hitoshi, Dixon Dan A., Oates John A., Boutaud Olivier. Tristetraprolin binds to the 3'-untranslated region of cyclooxygenase-2 mRNA. A polyadenylation variant in a cancer cell line lacks the binding site. J Biol Chem. 2003 Feb 10;278(16):13928–13935. doi: 10.1074/jbc.M300016200. [DOI] [PubMed] [Google Scholar]
  37. Sheng H., Shao J., Dubois R. N. K-Ras-mediated increase in cyclooxygenase 2 mRNA stability involves activation of the protein kinase B1. Cancer Res. 2001 Mar 15;61(6):2670–2675. [PubMed] [Google Scholar]
  38. Shim Jaekyung, Karin Michael. The control of mRNA stability in response to extracellular stimuli. Mol Cells. 2002 Dec 31;14(3):323–331. [PubMed] [Google Scholar]
  39. Snee Mark, Kidd Grahame J., Munro Trent P., Smith Ross. RNA trafficking and stabilization elements associate with multiple brain proteins. J Cell Sci. 2002 Dec 1;115(Pt 23):4661–4669. doi: 10.1242/jcs.00137. [DOI] [PubMed] [Google Scholar]
  40. Sokolowski M., Furneaux H., Schwartz S. The inhibitory activity of the AU-rich RNA element in the human papillomavirus type 1 late 3' untranslated region correlates with its affinity for the elav-like HuR protein. J Virol. 1999 Feb;73(2):1080–1091. doi: 10.1128/jvi.73.2.1080-1091.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Turini Marco E., DuBois Raymond N. Cyclooxygenase-2: a therapeutic target. Annu Rev Med. 2002;53:35–57. doi: 10.1146/annurev.med.53.082901.103952. [DOI] [PubMed] [Google Scholar]
  42. Vakalopoulou E., Schaack J., Shenk T. A 32-kilodalton protein binds to AU-rich domains in the 3' untranslated regions of rapidly degraded mRNAs. Mol Cell Biol. 1991 Jun;11(6):3355–3364. doi: 10.1128/mcb.11.6.3355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wait Robin, Miller Ingrid, Eberini Ivano, Cairoli Fausto, Veronesi Cristina, Battocchio Massimiliano, Gemeiner Manfred, Gianazza Elisabetta. Strategies for proteomics with incompletely characterized genomes: the proteome of Bos taurus serum. Electrophoresis. 2002 Sep;23(19):3418–3427. doi: 10.1002/1522-2683(200210)23:19<3418::AID-ELPS3418>3.0.CO;2-7. [DOI] [PubMed] [Google Scholar]
  44. Wilusz C. J., Wormington M., Peltz S. W. The cap-to-tail guide to mRNA turnover. Nat Rev Mol Cell Biol. 2001 Apr;2(4):237–246. doi: 10.1038/35067025. [DOI] [PubMed] [Google Scholar]
  45. Winzen R., Kracht M., Ritter B., Wilhelm A., Chen C. Y., Shyu A. B., Müller M., Gaestel M., Resch K., Holtmann H. The p38 MAP kinase pathway signals for cytokine-induced mRNA stabilization via MAP kinase-activated protein kinase 2 and an AU-rich region-targeted mechanism. EMBO J. 1999 Sep 15;18(18):4969–4980. doi: 10.1093/emboj/18.18.4969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Worthington Mark T., Pelo Jared W., Sachedina Muhammadreza A., Applegate Joan L., Arseneau Kristen O., Pizarro Theresa T. RNA binding properties of the AU-rich element-binding recombinant Nup475/TIS11/tristetraprolin protein. J Biol Chem. 2002 Sep 24;277(50):48558–48564. doi: 10.1074/jbc.M206505200. [DOI] [PubMed] [Google Scholar]
  47. Xu K., Robida A. M., Murphy T. J. Immediate-early MEK-1-dependent stabilization of rat smooth muscle cell cyclooxygenase-2 mRNA by Galpha(q)-coupled receptor signaling. J Biol Chem. 2000 Jul 28;275(30):23012–23019. doi: 10.1074/jbc.M001611200. [DOI] [PubMed] [Google Scholar]
  48. Zhang W., Wagner B. J., Ehrenman K., Schaefer A. W., DeMaria C. T., Crater D., DeHaven K., Long L., Brewer G. Purification, characterization, and cDNA cloning of an AU-rich element RNA-binding protein, AUF1. Mol Cell Biol. 1993 Dec;13(12):7652–7665. doi: 10.1128/mcb.13.12.7652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Zhang Z., Sheng H., Shao J., Beauchamp R. D., DuBois R. N. Posttranscriptional regulation of cyclooxygenase-2 in rat intestinal epithelial cells. Neoplasia. 2000 Nov-Dec;2(6):523–530. doi: 10.1038/sj.neo.7900117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Zubiaga A. M., Belasco J. G., Greenberg M. E. The nonamer UUAUUUAUU is the key AU-rich sequence motif that mediates mRNA degradation. Mol Cell Biol. 1995 Apr;15(4):2219–2230. doi: 10.1128/mcb.15.4.2219. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES