The mRNA poly(A)-binding protein: localization, abundance, and RNA-binding specificity.
The poly(A)-binding protein (PABP) binds to the messenger (mRNA) 3'-poly(A) tail found on most eukaryotic mRNAs and together with the poly(A) tail has been implicated in governing the stability and the translation of mRNA. In order to further understand the role of the PABP in these processes, we have undertaken a detailed analysis of the cellular localization, the abundance, and the RNA-binding properties of the human PABP (hPABP). We raised monoclonal antibodies against the 70-kDa hPABP and confocal immunofluorescence microscopy with these antibodies reveals that it is localized exclusively to the cytoplasm. The hPABP exhibits a very low turnover rate in these cells and quantitative immunoblotting experiments demonstrated that growing HeLa cells contain a surprisingly high number of approximately 8 x 10(6) PABP molecules per cell, which corresponds to an intracellular concentration of about 4 microM. In an in vitro selection/amplification assay from random sequence oligonucleotide pools the hPABP selects oligo(rA)-rich sequences and it binds oligo(rA)25 with an apparent Kd of 7 nM. The hPABP binds to unrelated RNA sequences with an about 100-fold lower affinity (Kd > or = 0.5 microM). The abundance of the hPABP indicates that there is an approximately three-fold excess of the protein over binding sites on cytoplasmic poly(A). This excess and the high concentration of the hPABP, which is three orders of magnitude above its Kd for oligo(rA)25, suggest that the hPABP may bind to additional, lower affinity binding sites in vivo.
Görlach
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Howard Hughes Medical Institute, University of Pennsylvania School of Medicine, Philadelphia 19104-6148.
Burd
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Dreyfuss
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D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D011061:Poly A / Q000235:genetics / Q000378:metabolism*; D039102:Poly(A)-Binding Proteins; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014162:Transfection
D000911:Antibodies, Monoclonal; D039102:Poly(A)-Binding Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D011061:Poly A