Alpha-globin expression: post transcriptional mechanisms

描述(由申请人提供)：珠蛋白mRNAs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。红系分化过程中珠蛋白mRNAs选择性稳定的基础尚不清楚。我们实验室正在使用人类α-珠蛋白mRNA作为研究这一问题的模型。在目前的资助期内进行的遗传、生化和体内表达研究表明，序列特异性的3‘UTRRNA-蛋白质(RNP)复合体(’α-复合体‘)在稳定α珠蛋白mRNA方面起着核心作用。富含C的结合基序的突变或阻断AlphaCP蛋白的结合使α-复合体失活，导致α-珠蛋白mRNA稳定性的逐渐丧失。这种稳定性的丧失可以通过人工将AlphaCP拴在3‘UTR上而完全恢复。α-CP在组织中广泛分布，这表明α-复合体的红系限制性作用是由对α-CP或相互作用的RNP组分的特定修饰决定的。主要的αCP异构体在胞核和胞浆中有不同的定位。证据表明，α-CP在α-珠蛋白mRNA稳定中的细胞质作用被参与促进α-珠蛋白mRNA加工的独立核功能(S)所补充。在拟议的研究中，将探索参与人α-珠蛋白mRNA选择性稳定的途径，以及在α-珠蛋白基因表达中α-CPs的核和细胞质功能之间的相互关系。目的I.确定介导α-珠蛋白信使核糖核酸稳定的α复合体的相互作用。目的II.明确α-珠蛋白基因稳定红系细胞的机制(S)以及α-珠蛋白基因如何在红系细胞中逃避衰变。目的III.确定α-CP如何促进α-珠蛋白转录本的核处理，以及这些核事件如何与α-CP介导的细胞质调控相结合。这些研究将扩展我们之前关于α-珠蛋白基因表达的工作，定义新的mRNA衰退途径，并建立一个在红系基因表达中协调核和细胞质转录后调控的范例。

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.