Functional antagonism between RNA binding proteins HuR and CUGBP2 determines the fate of COX-2 mRNA translation.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
CUGBP2 overrides HuR and suppresses COX-2 mRNA translation.
Sureban
Sripathi M
SM
Department of Medicine, University of Oklahoma Health Science Center, Oklahoma City, Oklahoma 73104, USA.
Murmu
Nabendu
N
Rodriguez
Pavel
P
May
Randal
R
Maheshwari
Reeti
R
Dieckgraefe
Brian K
BK
Houchen
Courtney W
CW
Anant
Shrikant
S
D020413:3' Untranslated Regions / Q000378:metabolism* / Q000528:radiation effects; D000818:Animals; D000954:Antigens, Surface / Q000235:genetics / Q000378:metabolism*; D001667:Binding, Competitive; D000067878:CELF Proteins; D002467:Cell Nucleus / Q000378:metabolism; D051546:Cyclooxygenase 2 / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D019281:Dimerization; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; D005720:Gamma Rays; D045325:HCT116 Cells; D019073:HT29 Cells; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D041681:NIH 3T3 Cells; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D014176:Protein Biosynthesis / Q000528:radiation effects; D025941:Protein Interaction Mapping / Q000379:methods; D021381:Protein Transport; D020871:RNA Stability; D012333:RNA, Messenger / Q000378:metabolism* / Q000528:radiation effects; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism; D014162:Transfection
D020413:3' Untranslated Regions; D000954:Antigens, Surface; D000067878:CELF Proteins; C117363:CELF2 protein, human; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; C098770:ELAVL1 protein, human; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human