MECHANISTIC BASIS FOR B-Globin mRNA Stability

Children'S Hospital Of Philadelphia, Philadelphia PA

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Abstract

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