Identification of Ebp1 as a component of cytoplasmic bcl-2 mRNP (messenger ribonucleoprotein particle) complexes.
The 3'-UTR (untranslated region) of bcl-2 mRNA contains an ARE (AU-rich element) that potentially regulates the stability of bcl-2 mRNA in a cell specific fashion. Previous studies have demonstrated that multiple proteins interact with bcl-2 mRNA in HL-60 (human leukaemia-60) cells, potentially contributing to the overexpression of Bcl-2 protein. Treatment of HL-60 cells with taxol or okadaic acid has been shown to induce destabilization of bcl-2 mRNA, which was associated with decreased binding of trans-acting factors to bcl-2 mRNA. Nucleolin has been identified as one of the bcl-2 mRNA-binding proteins [Sengupta, Bandyopadhyay, Fernandes and Spicer (2004) J. Biol. Chem. 279, 10855-10863]. In an effort to identify additional bcl-2 mRNA-binding proteins, two polypeptides of approx. 45 kDa and 60 kDa were isolated from HL-60 cells by ARE(bcl-2) (transcripts that contain bcl-2 AREs) RNA affinity chromatography. These proteins were identified as the human proliferation associated protein, Ebp1, and human DRBP76 (double stranded RNA-binding protein 76) respectively, by MALDI (matrix-assisted laser-desorption ionization)-MS. RNA electrophoretic mobility shift assays indicated that recombinant Ebp1 binds to ARE(bcl-2) RNA but not to the group 1 ARE present in GM-CSF (granulocyte macrophage-colony stimulating factor) mRNA in vitro. Antibody supershift assays demonstrated that Ebp1 is present in protein-ARE(bcl-2) RNA complexes formed with cytosolic HL-60 extracts. The interaction of Ebp1 with bcl-2 mRNA in HL-60 cells was also demonstrated by RNA co-immunoprecipitation assays. This interaction was not detected in extracts of taxol-treated HL-60 cells. Immunoprecipitation assays further revealed that Ebp1 co-precipitates with nucleolin from HL-60 cytoplasmic extracts. The observation that co-precipitation was decreased when extracts were treated with RNase suggests that Ebp1 and nucleolin are present in the same bcl-2 mRNP (messenger ribonucleoprotein particle) complexes. RNA-decay assays further demonstrated that Ebp1 decreased the rate of decay of beta-globin-ARE(bcl-2) transcripts in HL-60 cell extracts. Collectively, these results indicate a novel function for Ebp1 in contributing to the regulation of bcl-2 expression in HL-60 cells.
Bose
Sudeep K
SK
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 173 Ashley Avenue, P.O. Box 250509, Charleston, SC 29425, USA.
Sengupta
Tapas K
TK
Bandyopadhyay
Sumita
S
Spicer
Eleanor K
EK
D020413:3' Untranslated Regions / Q000235:genetics*; D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000032:analysis / Q000502:physiology*; D002846:Chromatography, Affinity; D003600:Cytosol / Q000737:chemistry; D019254:Genes, bcl-2; D005914:Globins / Q000235:genetics; D018922:HL-60 Cells / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D050779:Nuclear Factor 90 Proteins / Q000032:analysis; D019319:Okadaic Acid / Q000494:pharmacology; D017239:Paclitaxel / Q000494:pharmacology; D010750:Phosphoproteins / Q000502:physiology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism / Q000528:radiation effects; D016601:RNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000502:physiology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014466:Ultraviolet Rays; D000097796:Nucleolin
D020413:3' Untranslated Regions; D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D005914:Globins; D046911:Macromolecular Substances; D050779:Nuclear Factor 90 Proteins; D019319:Okadaic Acid; D017239:Paclitaxel; D010750:Phosphoproteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D000097796:Nucleolin; C429643:ILF3 protein, human; C101633:PA2G4 protein, human