Red Cell Band 4.1 Developmental Changes in RNA Splicing

描述(由申请人提供)：该项目的长期目标是描述在红细胞生成晚期控制替代前-mRNA剪接的调控网络的特征。潜在的假设是，RNA加工是基因表达的关键调节因素，随着细胞形态和功能的重塑，分化阶段特定剪接的“开关”改变了红系蛋白质的结构和功能。在蛋白4.1R前-mRNA中，外显子16(E16)在早期红系祖细胞中被跳过，但在晚期细胞中存在；这种剪接开关对血影蛋白-肌动蛋白结合和红细胞膜机械稳定性是必不可少的。初步研究已经在这些细胞中发现了另外两个剪接开关。4.1R Pre-mRNA的机制研究表明，由于剪接抑制因子hnRNP A1与E16中的沉默元件结合，E16在早期红细胞中受到抑制；转换是通过在晚期红细胞中A1表达减少而发生的。在其他系统中，受调控的选择性剪接通常是通过增强子和沉默蛋白之间的“动态拮抗”来调节的，它们竞争性地结合到RNA中的调控位点。初步研究已经在E16的3‘剪接位点区、嘌呤富集区和下游内含子中发现了增强子元件。新的数据表明，一种新的RNA结合/剪接因子Fox-2作用于内含子增强子，刺激El6剪接。为了了解多种调控信号如何整合来确定E16剪接，并将这些研究扩展到其他红系基因，提出了以下具体目标：(1)探索E16沉默和增强子的功能，重点是增强子的识别和与A1沉默的功能相互作用。(2)探讨内含子Fox-2剪接增强子的作用机制及其与其他E16剪接调控因子的相互作用。(3)通过鉴定和分析目前已知的三种剪接开关以外的新的红细胞剪接开关，以及表征剪接因子在红细胞中的表达模式，探索选择性剪接在红细胞生成中的更大作用。这些目标将利用RNA剪接分析和RNA：蛋白质结合方法，类似于已经应用于A1消音器分析的方法，并将利用最近的计算和微阵列技术进步。这些目标的实现将增加我们对红系细胞生理剪接开关的理解，并为新的FOX剪接增强剂的功能提供新的见解，这些剪接增强剂对后生动物的组织特异性剪接具有普遍重要性。许多人类疾病是由前mRNA剪接中的遗传异常引起的，包括增强子/沉默调控的缺陷；因此，了解红系剪接程序可能会有未来的治疗应用。

DESCRIPTION (provided by applicant): The long term objective of this project is to characterize the regulatory networks that control alternative pre-mRNA splicing in late erythropoiesis. The underlying hypothesis is that RNA processing is a critical regulator of gene expression, and that differentiation stage-specific splicing "switches" alter the structure and function of erythroid proteins as the cells are morphologically and functionally remodeled. In protein 4.1R pre-mRNA, exon 16 (E16) is skipped in early erythroid progenitors but included in later cells; this splicing switch is essential for spectrin-actin binding and red cell membrane mechanical stability. Preliminary studies have identified two additional splicing switches in these cells. Mechanistic studies with 4.1R pre-mRNA indicate that E16 is repressed in early erythroblasts due to binding of the splicing repressor, hnRNP A1, to silencer elements in E16; switching occurs via decreased A1 expression in late erythroblasts. In other systems, regulated alternative splicing is often mediated by "dynamic antagonism" between enhancer and silencer proteins binding competitively to regulatory sites in the RNA. Preliminary studies have identified enhancer elements in the 3' splice site region, the purine-rich region of E16, and the downstream intron. New data show that a novel RNA binding/splicing factor, Fox-2, acts at the intron enhancer to stimulate El6 splicing. To understand how multiple regulatory signals are integrated to determine E16 splicing, and to extend these studies to other erythroid genes, the following specific aims are proposed: (1) Explore E16 silencer and enhancer functions, focusing on enhancer identification and functional interactions with the A1 silencer. (2) Explore the mechanism of action of the intronic Fox-2 splicing enhancer, and its interactions with other E16 splicing regulators. (3) Explore the larger role of alternative splicing in erythropoiesis, by identifying/analyzing new erythroblast splicing switches besides the three currently known, and characterizing splicing factor expression patterns in erythroblasts. These aims will utilize RNA splicing assays and RNA:protein binding methods similar to those already applied to analysis of the A1 silencer, and will take advantage of recent computational and microarray technical advances. Achievement of these aims will increase our understanding of physiological splicing switches in erythroid cells, and provide new insights into function of novel Fox splicing enhancers of general importance to tissue-specific splicing in metazoan organisms. Many human diseases arise from genetic aberrations in pre-mRNA splicing, including defects in enhancer/silencer regulation; understanding the erythroid splicing program may thus have future therapeutic applications.