Globin gene expression is maintained and accentuated as a consequence of the unusual stability of the globin mRNAs during differentiation into a transcriptionally silent enucleated erythrocyte. Relatively little is known about the components that regulate globin mRNA stability. Irregularities in mRNA stability can have profound consequences that manifest as clinical phenotypes including thalassemias as exemplified by the athalassemia, a Constant Spring (a). Patients with the a S variant are severely anemic with virtually no mRNA in circulating reticulocytes due to a mutation that causes instability of the aCS mRNA. However, the molecular mechanisms involved in specific turnover of mRNAs are poorly understood and very few nucleases involved in the degradation of mRNA have been identified. We have devised an in vitro mRNA decay assay which recapitulates regulated mRNA turnover of the a-globin mRNA and have identified a sequence specific endoribonuclease, ErEN, which is involved in the degradation of this mRNA. ErEN is an erythroid specific endoribonuclease which specifically cleaves the a-globin mRNA both in vitro and in cells thereby demonstrating that this nuclease activity is essential for the normal biogenesis of a-globin mRNA. The long term objective of this proposal is to understand the determinants that regulate globin mRNA stability and decay. The focus of this proposal is:
(AIM 1) to biochemically isolate and molecularly clone ErEN;
(AIM 2) to functionally characterize the endoribonuclease activity and its expression profile in erythropoiesis;
(AIM 3) to initiate targeted regulation of heterologous mRNA and (AIM 4) to identify additional erythroid mRNAs regulated by ErEN. A thorough understanding of all the molecular controls of globin gene expression are necessary to efficiently ameliorate hemoglobinopathies and human genetic disorders in general. This work will provide fundamental insights into mRNA turnover, which is an important yet relatively unexplored component of gene expression, and afford novel approaches to regulate gene expression in therapeutic strategies.
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