ALPHA-GLOBIN EXPRESSION: POSTTRANSCRIPTIONAL MECHANISMS

描述：（研究者摘要）正常水平和发育对照 胎儿和成人红细胞珠蛋白合成的关键依赖于 编码mRNA的不寻常的稳定性。我们先前的研究建立了一种联系 人（h）α-珠蛋白mRNA的稳定化和序列的形成之间的关系 特异性RNA-蛋白质（RNP）复合物（"α-复合物"）。 我们假设这个由一个确定的多嘧啶束组成的a-复合体 由序列特异性RNA结合蛋白aCP结合，稳定α-珠蛋白mRNA 通过控制mRNA衰变中的一个或多个限速步骤。这些 在本建议中，将通过重点关注三个具体问题来扩大观察范围。 目标。在目标I中，我们专注于结构决定因素的表征， ha-珠蛋白mRNA稳定化的机制。使用一组Tet-transactivator 细胞系，我们将表征和比较α-珠蛋白mRNA衰变途径， 红细胞和非红细胞环境，识别和表征影响 的5'UTR和编码序列对a-Complex功能的影响，并确定aCP是否 完全足以介导Ha-珠蛋白mRNA的稳定。在Aim II中， 表征参与α-复合物作用的蛋白质-蛋白质相互作用。 将评估aCP与候选配偶体蛋白的相互作用，并将其应用于临床。 使用体外mRNA衰变测定测试功能重要性。在Aim M中，我们 将表征细胞核和细胞质aCP在以下方面的相应作用： a复合体的组装和功能。我们将确定aCP中的元素， 指示其亚细胞定位，确定aCP是否与 ha-珠蛋白mRNA在细胞核中的表达，决定细胞质aCP作用于 ha-globin mRNA，并设计aCP的显性负突变以探测其 不同的细胞核和细胞质功能。的表征 HA-珠蛋白mRNA稳定性的决定因素和机制 在这个建议中概述的是核心的珠蛋白基因的理解 健康和疾病的表达以及设计治疗方法， 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.