This proposal tests the hypothesis that Globin mRNAs contain determinant(s) critical to their stability and their consequent rapid accumulation in the developing erythrocyte. During a 3-5 day window of erythroid differentiation, globin mRNAs increase from less than 0.1% to greater than 95% of total cellular mRNA. This accumulation is dependent on both transcriptional activation of the globin genes and the unusual stability of the encoded globin mRNAs in erythroid cells. We plan to investigate the structural basis for the stability of globin mRNA. Our starting point will be to study a naturally occurring alpha-thalassemia mutation; alphaConstant Spring (alphaCS). Our preliminary data demonstrates that this mutation of the normal translation termination codon (UAA-->CAA) of the alpha2-globin mRNA results in its destabilization. Preliminary data further suggests that this destabilization is mechanistically linked to the entry of the translating ribosome into the 3' nontranslated region. We will study this destabilization of CS mRNA in detail and use the findings to suggest the position and mode of action of putative stability determinants in native alpha2-globin mRNA. The experimental approach will be based upon transfections of alpha2-globin genes (native and derivative) into tissue culture cells (erythroid and non erythroid) and studying the effect of informative mutations (including CS) on the stability of the encoded mRNAs. Preliminary results from site-specific mutations indicate that positive determinants of alpha2-globin mRNA stability reside in the 3' NTR. Potential interactions of these structural determinants with trans-acting factors will also be explored. The results of these studies will be generalized to other globin mRNAs and to nonglobin mRNAs whose message levels change during erythroid differentiation in order to determine whether there exist shared regulatory mechanisms of mRNA stability. Data from these experiments will contribute to a fundamental understanding of globin gene expression essential in the formulation of molecular genetic approaches to the therapy of sickle cell anemia.
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