Sickle cell anemia can be a devastating disease. Amelioration its severity have been approached at many levels. The most fundamental approach involves altering the pattern of globin gene expression and hemoglobin synthesis. Such an effort necessitates a full understanding of the genetic determinants of erythroid differentiation and function. It is evident that this is a complex, multifaceted process involving transcriptional and post-transcriptional controls of a large number of genes. Our particular interest is role of mRNA stability in this process. Although globin gene expression is initiated by transcriptional activation, the accumulation of globin mRNAs to greater than 95% of total cellular mRNA is dependent on their unusual stabilities. Mutations in human alpha-globin mRNA that destroy its stability result in loss of gene expression and consequent disease (alpha-thalassemia). Studies which have been completed during the first three years of the current renewal of the Comprehensive Sickle Cell Center Grant have demonstrated that stability of human alpha-globin mRNA depends on a defined cis determinant in the 3 'UTR. The stability determinant functions via assembly of a multicomponent RNA-protein (RNP) complex. One of the proteins in the alpha-complex is a 39 kD cytoplasmic RNA binding protein with a polyC binding-specificity. This protein is necessary, but not sufficient, for alpha-complex formation. In this competitive renewal we propose to continue our studies of globin mRNA stability with three Specific Aims: (1) Identify the full complement of proteins that compose the human alpha-complex. Studies the include biochemical purification, genetic screens, and RNA/protein crosslinking studies. (2) Characterize the higher-order structure of the alpha-complex. This will include determination of its overall mass, the stoichiometry of its protein components, and the nature of the RNA-protein interactions. (3) Characterize the rate limiting steps in alpha-globin mRNA turnover. Analyses will be carried out on the degradation patterns of normal alpha- globin mRNA and mutant alpha-globin mRNAs which cannot form the stability complex. The goal of this project is to extend the understanding of globin gene expression and generate new tools for its therapeutic modulation.
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