Programmed Changes in Alternative Splicing Within Erythr

描述（由申请人提供）：R21提案中启动的主要长期研究目的是表征红细胞选择性剪接程序，包括(a)鉴定红细胞选择性前mRNA剪接中的阶段特异性开关，以及(b)阐明主要剪接调节蛋白在影响该程序中的作用。假设选择性剪接程序在调节关键红系蛋白的结构中起主要作用，因此这些研究可能会刺激新的红系蛋白同种异构体的功能研究。先前的研究为这些概念的原理提供了证据，这些研究表明4.1R蛋白基因中选择性前mrna剪接的阶段特异性开关对红细胞膜的机械特性至关重要。以往对红系基因表达的全基因组分析已经为每个基因的表达水平提供了有价值的定量数据。然而，现有的定量每个基因mRNA输出的技术无法区分由选择性剪接产生的mRNA的质的差异，这可能完全改变基因的功能。在这个R21应用中，新型Affymetrix微阵列具有询问几乎所有已知和预测的外显子的能力，将用于研究红细胞生成过程中选择性剪接的程序性变化。使用阵列原型研究版本的初步合作实验证实，它们有效地识别了分化细胞类型之间的剪接差异。通过扩展，他们还应该检测单个谱系分化过程中剪接的阶段特异性变化；例如，以前所未有的分子分辨率促进红系转录组的检查。从培养的处于不同分化阶段的人和小鼠红系祖rna中制备探针，将其杂交到微阵列中，并分析外显子表达模式的变化。本文提出了两个目标：(1)全面分析人类和小鼠模型中构成正常红细胞选择性剪接程序的pre-mRNA剪接的阶段特异性开关；(2)通过测量rnai介导的剪接因子表达水平下调所发生的剪接变化，从功能上评估所选剪接因子蛋白在红细胞分化中的作用。这些目标的成功实现将为红系选择性剪接程序的范围提供重要的新见解；促进未来剪接开关机制的机理研究；并刺激红细胞细胞质、细胞骨架和膜蛋白新亚型的功能分析。在包括肌肉和大脑在内的其他组织中已经描述了剪接调节中的遗传干扰；本研究为发现红系细胞中新的剪接缺陷提供了基础。最终，模型系统中正在开发的治疗性剪接缺陷纠正方法可能也允许纠正红系细胞中的这种缺陷。

DESCRIPTION (provided by applicant): The major long range purpose of the studies initiated in this R21 proposal is to characterize the erythroid alternative splicing program, including (a) identification of erythroid stage-specific switches in alternative pre- mRNA splicing, and (b) elucidating the role of major splicing regulatory proteins in effecting this program. The alternative splicing program is hypothesized to play a major role in regulating structure of key erythroid proteins, and these studies may therefore stimulate functional studies of novel erythroid protein isoforms. Proof of principle for these concepts is provided by previous studies of a stage-specific switch in alternative pre-mRNA splicing in the protein 4.1R gene that is critical for erythroid membrane mechanical properties. Previous genome-wide analyses of erythroid gene expression have provided valuable quantitative data on the expression levels of each gene. However, existing technologies for quantitating mRNA output from each gene fail to distinguish qualitative differences in mRNA, generated by alternative splicing, which may completely alter gene function. In this R21 application, novel Affymetrix microarrays with the capacity to interrogate virtually all known and predicted exons will be used to study programmed changes in alternative splicing during erythropoiesis. Preliminary collaborative experiments using the prototype research version of the arrays confirm that they efficiently identify splicing differences between differentiated cell types. By extension, they should also detect stage-specific changes in splicing during differentiation of a single lineage; e.g., facilitating examination of the erythroid transcriptome with unprecedented molecular resolution. Probes prepared from cultured human and mouse erythroid progenitor RNAs at different stages of differentiation will be hybridized to the microarray and analyzed to elucidate changes in exon expression patterns. Two aims are proposed: (1) Perform a comprehensive analysis of stage-specific switches in pre-mRNA splicing that constitute the normal erythroid alternative splicing program in human and mouse models, and (2) Functionally assess the role of selected splicing factor proteins in erythroid differentiation by measuring splicing changes that occur in response to RNAi-mediated knockdown of splicing factor expression levels. Successful accomplishment of these goals should provide significant new insights into the scope of the erythroid alternative splicing program; facilitate future mechanistic studies of splicing switch mechanisms; and stimulate functional analyses of novel isoforms of erythroid cytoplasmic, cytoskeletal and membrane proteins. Genetic disturbances in splicing regulation have been described in other tissues including muscle and in brain; the present studies may provide a foundation for discovery of novel splicing defects in erythroid cells. Ultimately, methods for therapeutic correction of splicing defects, under development in model systems, may allow correction of such defects in erythroid cells as well.