Red Cell Band 4.1 Developmental Changes in RNA Splicing

描述（由申请人提供）：本项目的长期目标是表征控制晚期红细胞生成中前体mRNA可变剪接的调控网络。潜在的假设是RNA加工是基因表达的关键调节因子，并且分化阶段特异性剪接"开关"随着细胞的形态和功能重塑而改变红系蛋白的结构和功能。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，但在后期细胞中被包括;这种剪接开关对于血影蛋白-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确定了两个额外的剪接开关。4.1R前mRNA的机制研究表明，E16在早期成红细胞中由于剪接阻遏物hnRNP A1与E16中的沉默元件的结合而被抑制;通过晚期成红细胞中A1表达的降低而发生转换。在其他系统中，受调节的选择性剪接通常由竞争性结合RNA中的调节位点的增强子和沉默子蛋白之间的"动态拮抗作用"介导。初步研究已经确定了增强子元件在3 '剪接位点区域，嘌呤丰富的区域E16，和下游内含子。新的数据表明，一种新的RNA结合/剪接因子Fox-2在内含子增强子处起作用以刺激E16剪接。为了了解E16基因剪接过程中多种调控信号的相互作用，并将这些研究扩展到其他红系基因，本文提出了以下具体目标：（1）研究E16沉默子和增强子的功能，重点是增强子的鉴定以及与A1沉默子的功能相互作用。(2)探索内含子Fox-2剪接增强子的作用机制及其与其他E16剪接调节因子的相互作用。(3)探索选择性剪接在红细胞生成中的更大作用，通过鉴定/分析除了目前已知的三种剪接开关之外的新的成红细胞剪接开关，并表征成红细胞中的剪接因子表达模式。这些目标将利用RNA剪接分析和RNA：蛋白质结合方法类似于那些已经应用于分析的A1沉默，并将利用最近的计算和微阵列技术的进步。这些目标的实现将增加我们对红系细胞中生理剪接开关的理解，并为新型Fox剪接增强子对后生动物生物体中组织特异性剪接的重要性提供新的见解。许多人类疾病源于前体mRNA剪接中的遗传畸变，包括增强子/沉默子调节中的缺陷;因此，了解红细胞剪接程序可能具有未来的治疗应用。

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.