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).
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.
Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College London, 1 Aspenlea Road, Hammersmith, London W6 8LH, U.K.
Gareth
G
Sully
Jonathan L E
JL
Dean
Robin
R
Wait
Lesley
L
Rawlinson
Tomas
T
Santalucia
Jeremy
J
Saklatvala
Andrew R
AR
Clark
D020413:3' Untranslated Regions / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D000954:Antigens, Surface; D001482:Base Composition; D001483:Base Sequence; D045744:Cell Line, Tumor; D017124:Conserved Sequence / Q000235:genetics / Q000502:physiology*; D051546:Cyclooxygenase 2; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000235:genetics*; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; D024202:Electrophoretic Mobility Shift Assay; D006367:HeLa Cells; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics* / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics* / Q000502:physiology; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics* / Q000502:physiology; D020871:RNA Stability / Q000235:genetics / Q000502:physiology; D012334:RNA, Neoplasm / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D038621:Regulatory Sequences, Ribonucleic Acid / Q000235:genetics / Q000502:physiology*; D012097:Repressor Proteins / Q000235:genetics*; D013329:Structure-Activity Relationship; D051816:Tristetraprolin
D020413:3' Untranslated Regions; D000954:Antigens, Surface; D004268:DNA-Binding Proteins; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; C098770:ELAVL1 protein, human; C000630986:HNRNPD protein, human; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D; D017874:Immediate-Early Proteins; D007527:Isoenzymes; D008565:Membrane Proteins; D012334:RNA, Neoplasm; D016601:RNA-Binding Proteins; D038621:Regulatory Sequences, Ribonucleic Acid; D012097:Repressor Proteins; D051816:Tristetraprolin; C497552:ZFP36 protein, human; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases