Functional antagonism between RNA binding proteins HuR and CUGBP2 determines the fate of COX-2 mRNA translation.
Cyclooxygenase-2 (COX-2) expression is regulated at the levels of messenger RNA (mRNA) stability and translation by AU-rich elements (ARE) located in its 3' untranslated region (3'UTR). Although structurally homologous RNA binding proteins HuR and CUGBP2 stabilize COX-2 mRNA, HuR induces whereas CUGBP2 inhibits COX-2 mRNA translation. This study aimed to ... determine the antagonism between these proteins on COX-2 expression.COX-2 ARE binding activity was determined by nitrocellulose filter binding and UV cross-linking assays using recombinant glutathione S-transferase (GST)/HuR and GST/CUGBP2. Protein:protein interactions were determined by GST pull-down, yeast 2-hybrid, and immunocytochemistry assays. Nucleocytoplasmic shutting was determined by heterokaryon analyses. The effect of CUGBP2 and HuR on COX-2 ARE-dependent translation was shown by a chimeric luciferase mRNA containing COX-2 3'UTR. HT-29 cells were subjected to 12 Gy gamma-irradiation in a cesium irradiator.CUGBP2 and HuR bind with similar affinities to COX-2 ARE, but CUGBP2 competes with HuR for binding. In vitro, HuR and CUGBP2 heterodimerize. Furthermore, FLAG-tagged HuR and myc-tagged CUGBP2 colocalize in the nucleus of HCT-116 cells. Moreover, both proteins shuttled between the nucleus and cytoplasm. In vitro, HuR enhanced whereas CUGBP2 inhibited translation of the chimeric luciferase COX-2 3'UTR mRNA. Furthermore, CUGBP2 competitively inhibited HuR-mediated translation of the transcript. In HT-29 cells transfected with HuR and CUGBP2, a switch in COX-2 mRNA binding from predominantly HuR to CUGBP2 occurred after radiation treatment, which was coupled with increased silencing of the COX-2 mRNA.CUGBP2 overrides HuR and suppresses COX-2 mRNA translation.
Mesh Terms:
3' Untranslated Regions, Animals, Antigens, Surface, Binding, Competitive, Cell Nucleus, Cyclooxygenase 2, Cytoplasm, Dimerization, Gamma Rays, HCT116 Cells, HT29 Cells, Humans, Membrane Proteins, Mice, NIH 3T3 Cells, Nerve Tissue Proteins, Protein Binding, Protein Biosynthesis, Protein Interaction Mapping, Protein Transport, RNA Stability, RNA, Messenger, RNA-Binding Proteins, Recombinant Fusion Proteins, Repressor Proteins, Trans-Activators, Transfection
3' Untranslated Regions, Animals, Antigens, Surface, Binding, Competitive, Cell Nucleus, Cyclooxygenase 2, Cytoplasm, Dimerization, Gamma Rays, HCT116 Cells, HT29 Cells, Humans, Membrane Proteins, Mice, NIH 3T3 Cells, Nerve Tissue Proteins, Protein Binding, Protein Biosynthesis, Protein Interaction Mapping, Protein Transport, RNA Stability, RNA, Messenger, RNA-Binding Proteins, Recombinant Fusion Proteins, Repressor Proteins, Trans-Activators, Transfection
Gastroenterology
Date: Mar. 01, 2007
PubMed ID: 17383427
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