MECHANISTIC BASIS FOR B-Globin mRNA Stability

人β-珠蛋白的正常表达主要依赖于其编码mRNA的高稳定性。 尽管这一性质的重要性是明确的，相对较少的是知道的结构决定因素的β- 珠蛋白mRNA的稳定性或其中，有助于调节13-珠蛋白基因表达的方式。的 目前的建议旨在建立一个全面的了解的结构，因素， 影响人Ig-珠蛋白mRNA高稳定性的机制。这项研究是一项合作努力， 合作伙伴的机制和功能的方法，允许细胞培养和体外分析，以确认'在 有生理意义的动物模型。拟议的实验计划包括三个具体目标 旨在确认和扩展大量相关的初步数据。具体目标我会地图 在β-珠蛋白mRNA中的序列，这对它的高稳定性至关重要。的mRNA不稳定作用 将在培养的红系细胞中筛选覆盖3'UTR的突变，并研究它们的生理效应。 在完整的转基因动物中得到证实。具体目标II将确定反式作用细胞质因子， 与相关mRNA顺式元件相互作用。将使用纯化试剂盒从红系细胞裂解物中纯化因子。 Hover亲和色谱法，并使用常规技术鉴定。此外，细胞质 将直接测试已知稳定α-珠蛋白mRNA的因子对β-珠蛋白mRNA的作用 稳定特定目的HI将定义维持β-珠蛋白mRNA稳定性的机制。 纯化的反式作用因子的功能特性将使用已建立的体外实验来证明。 一种专门用于此目的的方法。相关的体外分析将定义初始和 在β-珠蛋白mRNA衰变的后续步骤中，特别注意poly（A）尾和 红细胞特异性核糖核酸内切酶的潜在作用。成功完成拟议的 研究将通过建立一个基本的 了解有助于Ig-珠蛋白mRNA稳定性的因素和机制，详细了解 这些基本过程将有助于设计新的治疗方法，改变13s和其他13样蛋白的表达。 球蛋白，包括由内源基因和转导基因编码的球蛋白，通过操纵 它们的同源mRNA

The normal expression of human beta-globin is crucially dependent upon the high stability of its encoding mRNA. Despite the clear importance of this property, relatively little is known about the structural determinants of beta- globin mRNA stability or the manner in which the,,' contribute to regulated 13-globin gene expression. The current proposal is designed to establish a comprehensive understanding of the structures, factors, and mechanisms that effect the high stability of human lg-globin mRNA. The research is a collaborative effort that partners mechanistic and functional approaches, permitting cell culture and in vitro analyses to be confirmed'in physiologically-meaningful animal models. The proposed experimental plan comprises three specific aims designed to confirm and extend a substantial body of relevant preliminary data. Specific Aim I will map sequences within the beta-globin mRNA that are crucial to its high stability. The mRNA-destabilizing effects of mutations blanketing the 3'UTR will be screened in cultured erythroid cells, and their physiological effects confirmed in intact transgenic animals. Specific Aim II will identify trans-acting cytoplasmic factors that interact with relevant mRNA cis-elements. The factor(s) will be purified from erythroid cell lysate using a hover affinity chromatographic method, and identified using conventional techniques. In addition, cytoplasmic factors that are known to stabilize ct-globin mRNA will be directly tested for their effect on beta-globin mRNA stability. Specific Aim HI will define the mechanism(s) through which beta-globin mRNA stability is maintained. The functional properties of the purified trans-acting factors will be demonstrated using an established in vitro method that wiII be specifically adapted for this purpose. Related in vitro analyses will define the initial and subsequent steps in beta-globin mRNA decay, with special atten:ion to the importance of the poly(A) tail and the potential role of an erythroid cell-specific endoribonuclease. The successful completion of the proposed research will provide new insights into the control of ig-globin gene expression by establishing a fundamental understanding of the factors and mechanisms that contribute to lg-globin mRNA stability, A detailed knowledge of these basic processes will facilitate design of novel therapies that modify expression of 13s and other 13-like globins, including globins encoded by both endogenous and transduced genes, by manipulating the stabilities of their cognate mRNAs.