Assembly of the alpha-globin mRNA stability complex reflects binary interaction between the pyrimidine-rich 3 ' untranslated region determinant and poly(C) binding protein alpha CP

Globin mRNAs accumulate to 95% of total cellular mRNA during terminal eryth roid differentiation, reflecting their extraordinary stability. The stabili ty of human alpha-globin mRNA is paralleled by formation of a sequence-spec ific RNA-protein (RNP) complex at a pyrimidine-rich site within its 3' untr anslated region (3'UTR), the alpha-complex. The proteins of the alpha-compl ex are widely expressed. The alpha-complex or a closely related complex als o 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 alpha CPs, members of a family of hnRNP K-homo logy domain poly(C) binding proteins, are essential constituents of the alp ha-complex. The ability of alpha CPs to homodimerize and their reported ass ociation with additional RNA binding proteins such as AU-rich binding facto r 1 (AUF1) and hnRNP K have suggested that the alpha-complex is a multisubu nit structure. In the present study, we have addressed the composition of t he alpha-complex. An RNA titration recruitment assay revealed that alpha CP s were quantitatively incorporated into the alpha-complex in the absence of associated AUF1 and hnRNP K. A high-affinity direct interaction between ea ch of the three major alpha CP isoforms and the alpha-globin 3'UTR was dete cted, suggesting that each of these proteins might be sufficient for alpha- complex assembly. This sufficiency was further supported by the sequence-sp ecific binding of recombinant alpha CPs to a spectrum of RNA targets. Final ly, density sedimentation analysis demonstrated that the alpha-complex coul d accommodate only a single alpha CP. These data established that a single alpha CP molecule binds directly to the alpha-globin 3'UTR, resulting in a simple binary structure for the alpha-complex.