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.
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3' Untranslated Regions, Antigens, Surface, Base Composition, Base Sequence, Cell Line, Tumor, Conserved Sequence, Cyclooxygenase 2, DNA Mutational Analysis, DNA-Binding Proteins, ELAV Proteins, ELAV-Like Protein 1, Electrophoretic Mobility Shift Assay, HeLa Cells, Heterogeneous-Nuclear Ribonucleoprotein D, Humans, Immediate-Early Proteins, Isoenzymes, Membrane Proteins, Molecular Sequence Data, Prostaglandin-Endoperoxide Synthases, RNA Stability, RNA, Neoplasm, RNA-Binding Proteins, Regulatory Sequences, Ribonucleic Acid, Repressor Proteins, Structure-Activity Relationship, Tristetraprolin