High-level stability of globin mRNAs is a major determinant of hemoglobin synthesis and erythrocyte function. The basis for selective stabilization of globin mRNAs during erythroid differentiation remains poorly understood. Our laboratory is using human alpha-globin mRNA as a model for the study of this problem. Genetic, biochemical, and in vivo expression studies carried out over the present funding period point to a central role for a sequence-specific 3'UTR RNA-protein (RNP) complex ('alpha- complex') in stabilizing alpha globin mRNA. Inactivation of the alpha-complex by mutation of the C-rich binding motif or by blocking the binding of the alphaCP protein results in an incremental loss of alpha-globin mRNA stability. This loss of stability can be fully restored by artificially tethering alphaCP to the 3'UTR. AlphaCPs are broadly distributed in tissues, suggesting that an erythroid- restricted role of the a-complex is dictated by specific modifications to alphaCP or to interacting RNP components. The major alphaCP isoforms are differentially localized in the nucleus and cytoplasm. Evidence suggests that the cytoplasmic role of alphaCPs in alpha-globin mRNA stabilization is complemented by separate nuclear function(s) involved in enhancement of alpha-globin mRNA processing. The pathways involved in selective stabilization of human alpha-globin mRNA and the interrelationships between nuclear and cytoplasmic functions of alphaCPs in alpha-globin gene expression will be explored in the proposed studies.
Aim I. Identify interactions at the alpha complex that mediate alpha-globin mRNA stabilization.
Aim II. Define the mechanism(s) of alpha-globin mRNA stabilization and how alpha-globin mRNA evades decay in erythroid cells.
Aim III. Determine how alphaCPs enhance nuclear processing of alpha-globin transcripts and how these nuclear events integrate with alphaCP-mediated cytoplasmic controls. These studies will extend our prior work on alpha-globin gene expression, define novel pathways of mRNA decay, and establish a paradigm for coordinated nuclear and cytoplasmic post-transcriptional controls in erythroid gene expression. ? ?
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