ALPHA-GLOBIN EXPRESSION: POSTTRANSCRIPTIONAL MECHANISMS

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DESCRIPTION: (Investigator's abstract) Normal levels and developmental control of globin synthesis in fetal and adult erythrocytes is critically dependent on the unusual stability of the encoding mRNAs. Our prior studies establish a link between stabilization of human (h)a-globin mRNA and formation of a sequence specific RNA-protein (RNP) complex ('a-Complex') at its 3' untranslated region. We hypothesize that this a-Complex, composed of a defined polypyrimidine tract bound by a sequence-specific RNA binding protein, aCP, stabilizes a-globin mRNA by controlling one or more rate-limiting steps in mRNA decay. These observations will be extended in this proposal by focusing on three Specific Aims. In Aim I we focus on characterization of structural determinants and mechanism(s) of ha-globin mRNA stabilization. Using a set of Tet-transactivator cell lines we will characterize and compare a-globin mRNA decay pathways in erythroid and nonerythroid environments, identify and characterize influences of 5'UTR and coding sequences on a-Complex function, and determine whether aCP is fully sufficient to mediate ha-globin mRNA stabilization. In Aim II we will characterize protein-protein interactions involved in a-Complex action. Interactions of aCP with candidate partner proteins will be assessed and their functional importance tested using an in vitro mRNA decay assay. In Aim m we will characterize the corresponding roles of nuclear and cytoplasmic aCP in assembly and function of the a-Complex. We will identify elements in aCP that dictate its subcellular localization, determine whether aCP associates with ha-globin mRNA in the nucleus, determine the site of cytoplasmic aCP action on ha-globin mRNA, and design dominant-negative mutations of aCP to probe its distinct nuclear and cytoplasmic functions. The characterization of the determinants and mechanisms involved in stabilization of ha-globin mRNA outlined in this proposal is central to a understanding of globin gene expression in health and disease and to the design of therapeutic approaches to a broad spectrum of hereditary anemias.