Abstract
Mammalian apolipoprotein B (apoB) mRNA editing is mediated by a multicomponent holoenzyme containing apobec-1 and ACF. We have now identified CUGBP2, a 54-kDa RNA-binding protein, as a component of this holoenzyme. CUGBP2 and ACF co-fractionate in bovine liver S-100 extracts, and addition of recombinant apobec-1 leads to assembly of a holoenzyme. Immunodepletion of CUGBP2 co-precipitates ACF, and these proteins co-localize the nucleus of transfected cells, suggesting that CUGBP2 and ACF are bound in vivo. CUGBP2 binds apoB RNA, specifically an AU-rich sequence located immediately upstream of the edited cytidine. ApoB RNA from McA cells, bound to CUGBP2, was more extensively edited than the unbound fraction. However, addition of recombinant CUGBP2 to a reconstituted system demonstrated a dose-dependent inhibition of C to U RNA editing, which was rescued with either apobec-1 or ACF. Antisense CUGBP2 knockout increased endogenous apoB RNA editing, whereas antisense knockout of either apobec-1 or ACF expression eliminated apoB RNA editing, establishing the absolute requirement of these components of the core enzyme. These data suggest that CUGBP2 plays a role in apoB mRNA editing by forming a regulatory complex with the three components of the minimal editing enzyme, apobec-1, ACF, and apoB RNA.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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APOBEC-1 Deaminase
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Active Transport, Cell Nucleus
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Animals
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Apolipoproteins B / metabolism*
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Base Sequence
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Blotting, Western
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CELF1 Protein
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Cattle
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Cell Line
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Cell Nucleus / metabolism
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Cloning, Molecular
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Cytidine Deaminase*
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Cytoplasm / metabolism
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DNA, Complementary / metabolism
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Dose-Response Relationship, Drug
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Glutathione Transferase / metabolism
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Holoenzymes / chemistry
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Liver / metabolism
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Microscopy, Fluorescence
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Molecular Sequence Data
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Oligonucleotides, Antisense / pharmacology
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Precipitin Tests
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Protein Binding
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RNA / metabolism
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RNA Editing*
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RNA Splicing
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RNA, Messenger / metabolism*
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RNA-Binding Proteins / genetics*
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RNA-Binding Proteins / metabolism*
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Rats
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Recombinant Fusion Proteins / metabolism
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Recombinant Proteins / metabolism
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Ribonucleoproteins / genetics*
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Ribonucleoproteins / metabolism*
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S100 Proteins / metabolism
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Subcellular Fractions / metabolism
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Transfection
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Transgenes
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Tumor Cells, Cultured
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Ultraviolet Rays
Substances
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Apolipoproteins B
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CELF1 Protein
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CELF1 protein, human
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DNA, Complementary
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Holoenzymes
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Oligonucleotides, Antisense
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RNA, Messenger
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RNA-Binding Proteins
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Recombinant Fusion Proteins
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Recombinant Proteins
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Ribonucleoproteins
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S100 Proteins
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RNA
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Glutathione Transferase
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APOBEC-1 Deaminase
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APOBEC1 protein, human
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Apobec1 protein, rat
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Cytidine Deaminase