ALPHA-GLOBIN EXPRESSION: POSTTRANSCRIPTIONAL MECHANISMS

描述：(研究人员摘要)正常水平和发育控制 胎儿和成人红细胞中的珠蛋白合成严重依赖于 编码mRNA的不同寻常的稳定性。我们之前的研究建立了一个联系 人(H)a-珠蛋白mRNA的稳定与序列形成之间的关系 位于其3‘非翻译区的特异性RNA-蛋白质(RNP)复合体(’a-复合体‘)。 我们假设这个α-复合体，由一个定义的多嘧啶区域组成 被序列特异性RNA结合蛋白ACP结合，稳定a-珠蛋白mRNA 通过控制信使核糖核酸衰变中的一个或多个限速步骤。这些 在本提案中，将通过重点关注三个具体的项目来扩展观察结果 目标。在Aim I中，我们重点描述结构决定因素和 HA-珠蛋白基因稳定机制(S)。使用一套Tet反式激活剂 我们将描述和比较细胞系中a-珠蛋白mRNA的衰退途径 红系和非红系环境，识别和表征影响 5‘非翻译区和编码序列的一个复数函数，并确定ACP 完全足以介导HA-珠蛋白mRNA的稳定。在AIM II中，我们将 描述参与α-复合体作用的蛋白质之间的相互作用。 将评估ACP与候选伙伴蛋白的相互作用，并对其 用体外信使核糖核酸衰变试验检测其功能重要性。在我们的目标中 将表征细胞核和细胞质ACP在 α-复合体的组装和功能。我们将确定机场核心计划中的元素 口述其亚细胞定位，确定ACP是否与 HA-珠蛋白在胞核中的表达，确定ACP在胞浆中的作用部位 HA-珠蛋白基因，设计ACP显性-负性突变检测其 不同的核质功能。《红楼梦》的特点 影响HA-珠蛋白mRNA稳定性的决定因素及机制 这一提案中概述的是理解珠蛋白基因的核心 在健康和疾病中的表达以及治疗方法的设计 广泛的遗传性贫血。

DESCRIPTION: (Investigator's abstract) Normal levels and developmental control of globin synthesis in fetal and adult erythrocytes is critically dependent on the unusual stability of the encoding mRNAs. Our prior studies establish a link between stabilization of human (h)a-globin mRNA and formation of a sequence specific RNA-protein (RNP) complex ('a-Complex') at its 3' untranslated region. We hypothesize that this a-Complex, composed of a defined polypyrimidine tract bound by a sequence-specific RNA binding protein, aCP, stabilizes a-globin mRNA by controlling one or more rate-limiting steps in mRNA decay. These observations will be extended in this proposal by focusing on three Specific Aims. In Aim I we focus on characterization of structural determinants and mechanism(s) of ha-globin mRNA stabilization. Using a set of Tet-transactivator cell lines we will characterize and compare a-globin mRNA decay pathways in erythroid and nonerythroid environments, identify and characterize influences of 5'UTR and coding sequences on a-Complex function, and determine whether aCP is fully sufficient to mediate ha-globin mRNA stabilization. In Aim II we will characterize protein-protein interactions involved in a-Complex action. Interactions of aCP with candidate partner proteins will be assessed and their functional importance tested using an in vitro mRNA decay assay. In Aim m we will characterize the corresponding roles of nuclear and cytoplasmic aCP in assembly and function of the a-Complex. We will identify elements in aCP that dictate its subcellular localization, determine whether aCP associates with ha-globin mRNA in the nucleus, determine the site of cytoplasmic aCP action on ha-globin mRNA, and design dominant-negative mutations of aCP to probe its distinct nuclear and cytoplasmic functions. The characterization of the determinants and mechanisms involved in stabilization of ha-globin mRNA outlined in this proposal is central to a understanding of globin gene expression in health and disease and to the design of therapeutic approaches to a broad spectrum of hereditary anemias.