Alpha-globin expression: post transcriptional mechanisms

描述（由申请人提供）：珠蛋白mrna的高水平稳定性是血红蛋白合成和红细胞功能的主要决定因素。红细胞分化过程中珠蛋白mrna选择性稳定的基础仍然知之甚少。我们的实验室正在使用人类α -珠蛋白mRNA作为模型来研究这个问题。在目前的资助期内进行的遗传、生化和体内表达研究表明，序列特异性3'UTR rna -蛋白（RNP）复合物（‘ α -复合物’）在稳定α珠蛋白mRNA方面发挥着核心作用。通过富c结合基序的突变或阻断α - acp蛋白的结合使α -复合物失活，导致α -珠蛋白mRNA稳定性的逐渐丧失。这种稳定性的损失可以通过人为地将alphap拴在3'UTR上来完全恢复。alphaCP广泛分布于组织中，这表明a-复合物的红系限制性作用是由对alphaCP或相互作用的RNP组分的特异性修饰决定的。主要的α - acp异构体在细胞核和细胞质中有不同的定位。有证据表明，α acps在稳定α -珠蛋白mRNA中的细胞质作用是由参与增强α -珠蛋白mRNA加工的单独核功能补充的。人类α -珠蛋白mRNA选择性稳定的途径以及α -珠蛋白基因表达中α acps的核和细胞质功能之间的相互关系将在拟议的研究中进行探索。目的1：确定α复合物中介导α珠蛋白mRNA稳定的相互作用。目的二世。确定α -珠蛋白mRNA稳定的机制以及α -珠蛋白mRNA如何在红细胞中逃避衰变。第三目标。确定α - acp如何增强α -珠蛋白转录本的核加工，以及这些核事件如何与α - acp介导的细胞质控制相结合。这些研究将扩展我们之前关于α -珠蛋白基因表达的工作，定义mRNA衰变的新途径，并建立红细胞基因表达中核和细胞质转录后协调控制的范例。

DESCRIPTION (provided by applicant): 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.