title: Assembly of the alpha-globin mRNA stability complex reflects binary interaction between the pyrimidine-rich 3' untranslated region determinant and poly(C) binding protein alphaCP.
abstract: Globin mRNAs accumulate to 95% of total cellular mRNA during terminal erythroid differentiation, reflecting their extraordinary stability. The stability of human alpha-globin mRNA is paralleled by formation of a sequence-specific RNA-protein (RNP) complex at a pyrimidine-rich site within its 3' untranslated region (3'UTR), the alpha-complex. The proteins of the alpha-complex are widely expressed. The alpha-complex or a closely related complex also assembles at pyrimidine-rich 3'UTR segments of other stable mRNAs. These data suggest that the alpha-complex may constitute a general determinant of mRNA stability. One or more alphaCPs, members of a family of hnRNP K-homology domain poly(C) binding proteins, are essential constituents of the alpha-complex. The ability of alphaCPs to homodimerize and their reported association with additional RNA binding proteins such as AU-rich binding factor 1 (AUF1) and hnRNP K have suggested that the alpha-complex is a multisubunit structure. In the present study, we have addressed the composition of the alpha-complex. An RNA titration recruitment assay revealed that alphaCPs were quantitatively incorporated into the alpha-complex in the absence of associated AUF1 and hnRNP K. A high-affinity direct interaction between each of the three major alphaCP isoforms and the alpha-globin 3'UTR was detected, suggesting that each of these proteins might be sufficient for alpha-complex assembly. This sufficiency was further supported by the sequence-specific binding of recombinant alphaCPs to a spectrum of RNA targets. Finally, density sedimentation analysis demonstrated that the alpha-complex could accommodate only a single alphaCP. These data established that a single alphaCP molecule binds directly to the alpha-globin 3'UTR, resulting in a simple binary structure for the alpha-complex.
authors.affiliation: Howard Hughes Medical Institute and Departments of Genetics and Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.
authors.forename: A N
authors.initials: AN
authors.lastname: Chkheidze
authors.forename: D L
authors.initials: DL
authors.lastname: Lyakhov
authors.forename: A V
authors.initials: AV
authors.lastname: Makeyev
authors.forename: J
authors.initials: J
authors.lastname: Morales
authors.forename: J
authors.initials: J
authors.lastname: Kong
authors.forename: S A
authors.initials: SA
authors.lastname: Liebhaber
mesh_terms: D020413:3' Untranslated Regions / Q000378:metabolism*; D003600:Cytosol; D004268:DNA-Binding Proteins; D005914:Globins / Q000235:genetics*; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D; D034641:Heterogeneous-Nuclear Ribonucleoprotein K; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D020014:K562 Cells; D011743:Pyrimidines / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000378:metabolism; D014157:Transcription Factors
chemical_list: D020413:3' Untranslated Regions; D004268:DNA-Binding Proteins; C000630986:HNRNPD protein, human; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D; D034641:Heterogeneous-Nuclear Ribonucleoprotein K; D034441:Heterogeneous-Nuclear Ribonucleoproteins; C094604:PCBP1 protein, human; D011743:Pyrimidines; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D014157:Transcription Factors; D005914:Globins