MECHANISTIC BASIS FOR B-Globin mRNA Stability

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The normal expression of human beta-globin is crucially dependent upon the high stability of its encoding mRNA. Despite the clear importance of this property, relatively little is known about the structural determinants of beta- globin mRNA stability or the manner in which the,,' contribute to regulated 13-globin gene expression. The current proposal is designed to establish a comprehensive understanding of the structures, factors, and mechanisms that effect the high stability of human lg-globin mRNA. The research is a collaborative effort that partners mechanistic and functional approaches, permitting cell culture and in vitro analyses to be confirmed'in physiologically-meaningful animal models. The proposed experimental plan comprises three specific aims designed to confirm and extend a substantial body of relevant preliminary data. Specific Aim I will map sequences within the beta-globin mRNA that are crucial to its high stability. The mRNA-destabilizing effects of mutations blanketing the 3'UTR will be screened in cultured erythroid cells, and their physiological effects confirmed in intact transgenic animals. Specific Aim II will identify trans-acting cytoplasmic factors that interact with relevant mRNA cis-elements. The factor(s) will be purified from erythroid cell lysate using a hover affinity chromatographic method, and identified using conventional techniques. In addition, cytoplasmic factors that are known to stabilize ct-globin mRNA will be directly tested for their effect on beta-globin mRNA stability. Specific Aim HI will define the mechanism(s) through which beta-globin mRNA stability is maintained. The functional properties of the purified trans-acting factors will be demonstrated using an established in vitro method that wiII be specifically adapted for this purpose. Related in vitro analyses will define the initial and subsequent steps in beta-globin mRNA decay, with special atten:ion to the importance of the poly(A) tail and the potential role of an erythroid cell-specific endoribonuclease. The successful completion of the proposed research will provide new insights into the control of ig-globin gene expression by establishing a fundamental understanding of the factors and mechanisms that contribute to lg-globin mRNA stability, A detailed knowledge of these basic processes will facilitate design of novel therapies that modify expression of 13s and other 13-like globins, including globins encoded by both endogenous and transduced genes, by manipulating the stabilities of their cognate mRNAs.