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)

• Published in: Biochemical journal Vol. 377; no. Pt 3; pp. 629 - 639
• Main Authors: Sully, Gareth, Dean, Jonathan L E, Wait, Robin, Rawlinson, Lesley, Santalucia, Tomas, Saklatvala, Jeremy, Clark, Andrew R
• Format: Journal Article
• Language: English
• Published: England 01.02.2004
• Subjects: 3' Untranslated Regions - genetics; 3' Untranslated Regions - metabolism; 3' Untranslated Regions - physiology; Antigens, Surface; Base Composition; Base Sequence; Cell Line, Tumor; Conserved Sequence - genetics; Conserved Sequence - physiology; Cyclooxygenase 2; DNA Mutational Analysis; DNA-Binding Proteins - genetics; ELAV Proteins; ELAV-Like Protein 1; Electrophoretic Mobility Shift Assay; HeLa Cells; Heterogeneous-Nuclear Ribonucleoprotein D - genetics; Heterogeneous-Nuclear Ribonucleoprotein D - metabolism; Humans; Immediate-Early Proteins - genetics; Immediate-Early Proteins - metabolism; Isoenzymes - genetics; Isoenzymes - physiology; Membrane Proteins; Molecular Sequence Data; Prostaglandin-Endoperoxide Synthases - genetics; Prostaglandin-Endoperoxide Synthases - physiology; Regulatory Sequences, Ribonucleic Acid - genetics; Regulatory Sequences, Ribonucleic Acid - physiology; Repressor Proteins - genetics; RNA Stability - genetics; RNA Stability - physiology; RNA, Neoplasm - genetics; RNA, Neoplasm - metabolism; RNA, Neoplasm - physiology; RNA-Binding Proteins - genetics; RNA-Binding Proteins - metabolism; RNA-Binding Proteins - physiology; Structure-Activity Relationship; Tristetraprolin
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj20031484

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.