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表征红细胞生成后期控制替代前mrna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，分化阶段特异性剪接“开关”在细胞形态学和功能重塑时改变红系蛋白的结构和功能。在蛋白4.1R pre-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含；这种剪接开关对于谱蛋白-肌动蛋白结合和红细胞的机械稳定性至关重要。初步研究已经在这些细胞中发现了两个额外的剪接开关。4.1R pre-mRNA的机制研究表明，E16在早期红母细胞中受到抑制是由于剪接抑制因子hnRNP A1与E16中的沉默元件结合；转换发生在晚期红母细胞A1表达的降低。在其他系统中，受调节的选择性剪接通常是由增强子和沉默子蛋白之间的“动态拮抗”介导的，它们竞争性地结合到RNA中的调节位点上。初步研究已经在3'剪接位点区域、E16富含嘌呤的区域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在内含子增强子处起作用，刺激El6剪接。为了了解多种调控信号如何整合决定E16剪接，并将这些研究扩展到其他红系基因，我们提出了以下具体目标：(1)探索E16沉默子和增强子的功能，重点研究增强子的鉴定和与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，以及它与其他E16剪接调节子的相互作用。(3)通过鉴定/分析除目前已知的三个红母细胞剪接开关外的新的红母细胞剪接开关，并表征红母细胞剪接因子的表达模式，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剪接测定和RNA：蛋白质结合方法，类似于那些已经应用于A1消音器分析的方法，并将利用最近的计算和微阵列技术的进步。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，并为对后生动物组织特异性剪接具有重要意义的新型Fox剪接增强子的功能提供新的见解。许多人类疾病是由前mrna剪接的遗传畸变引起的，包括增强子/沉默子调控的缺陷；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.