Abstract
Expression of the cyclin-dependent kinase inhibitor p21 is highly induced by many stresses, including exposure to short-wavelength UV light (UVC), which increases p21 mRNA stability. Investigation into the mechanisms underlying this stabilization process revealed that proteins present in cytoplasmic lysates of human RKO colorectal carcinoma cells formed complexes with p21 mRNA that were inducible by treatment with UVC and other stress agents. The ubiquitous Elav-type RNA-binding protein HuR was identified within the p21 mRNA-protein complexes, as antibodies recognizing HuR supershifted these complexes and revealed HuR-immunoreactive proteins complexing with p21 mRNA on Western blots. Lowering of endogenous HuR levels through expression of antisense HuR decreased p21 RNA-protein complexes, greatly reduced the UVC inducibility and half-life of p21 mRNA, and prevented UVC-mediated induction of luciferase activity in p21 3' untranslated region-containing reporter constructs. Our findings indicate that HuR plays a major role in regulating stress-induced p21 expression by enhancing p21 mRNA stability and that these effects are coupled to HuR's elevated presence in the cytoplasm.
MeSH terms
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Antigens, Surface*
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Blotting, Western
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Cell Nucleus / metabolism
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Colorectal Neoplasms
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Cyclin-Dependent Kinase Inhibitor p21
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Cyclins / genetics*
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Cyclins / isolation & purification
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Cyclins / metabolism*
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Cytoplasm / metabolism
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Dactinomycin / pharmacology
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ELAV Proteins
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ELAV-Like Protein 1
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Enzyme Inhibitors / metabolism
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Gene Expression Regulation, Neoplastic / drug effects
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Gene Expression Regulation, Neoplastic / physiology
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Gene Expression Regulation, Neoplastic / radiation effects*
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Green Fluorescent Proteins
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Humans
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Hydrogen Peroxide / pharmacology
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Luminescent Proteins / genetics
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Methyl Methanesulfonate / pharmacology
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Polymerase Chain Reaction
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RNA, Messenger / genetics
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RNA, Messenger / radiation effects
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RNA-Binding Proteins / genetics
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RNA-Binding Proteins / isolation & purification
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RNA-Binding Proteins / metabolism*
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Signal Transduction
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Transcription, Genetic / drug effects
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Transcription, Genetic / radiation effects
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Transfection
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Tumor Cells, Cultured
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Ultraviolet Rays*
Substances
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Antigens, Surface
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CDKN1A protein, human
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Cyclin-Dependent Kinase Inhibitor p21
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Cyclins
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ELAV Proteins
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ELAV-Like Protein 1
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ELAVL1 protein, human
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Enzyme Inhibitors
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Luminescent Proteins
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RNA, Messenger
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RNA-Binding Proteins
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Green Fluorescent Proteins
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Dactinomycin
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Methyl Methanesulfonate
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Hydrogen Peroxide