Programmed Changes in Alternative Splicing Within the Erythroid Transcriptome

红细胞转录组内选择性剪接的程序化变化

• 批准号: 7268079
• 负责人: JOHN G CONBOY
• 金额: $ 24.89万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7268079
• 关键词: Actins; Alternative Splicing; Binding; Biological Assay; Biological Models; Biology; Biotin; Brain; Brain Pathology; Cell Line; Cells; Code; Complex; Coupled; Cytoskeletal Proteins; Data; Defect; Development; Down-Regulation; Erythroblasts; Erythroid; Erythroid Cells; Erythropoiesis; Event; Exclusion; Exons; Foundations; Future; Gene Expression; Gene Expression Profile; Genes; Genetic; Genome; Goals; Grant; Heterogeneous Nuclear RNA; Human; Human Genome; Knockout Mice; Label; Laboratories; Measures; Mechanics; Mediating; Membrane; Membrane Proteins; Messenger RNA; Methods; Modeling; Molecular; Monitor; Mus; Muscle; Output; Pattern; Phycoerythrin; Plant Roots; Play; Procedures; Property; Protein Isoforms; Proteins; Protocols documentation; Purpose; RNA Interference; RNA Splicing; RNA-Binding Proteins; Range; Regulation; Relative (related person); Research; Research Personnel; Resolution; Resources; Role; Scanning; Specificity; Spectrin; Staging; Staining method; Stains; Streptavidin; Structure; System; Technology; Therapeutic; Time; Tissue-Specific Splicing; Tissues; Transcript; base; cDNA Probes; cell type; computerized data processing; concept; design; erythroid differentiation; gene function; genetic regulatory protein; hnRNP A1; insight; mRNA Precursor; mouse model; novel; progenitor; programs; protein 4.1; prototype; research study; response; tool; transcription factor

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.

描述（由申请人提供）：本 R21 提案中启动的研究的主要长期目的是表征红细胞选择性剪接程序，包括 (a) 鉴定选择性前 mRNA 剪接中的红细胞阶段特异性开关，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细胞蛋白的结构中发挥重要作用，因此这些研究可能会刺激新型红细胞蛋白亚型的功能研究。先前对蛋白质 4.1R 基因中选择性前 mRNA 剪接的阶段特异性转换的研究提供了这些概念的原理证明，这对于红细胞膜的机械特性至关重要。先前对红系基因表达的全基因组分析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的 mRNA 输出的技术无法区分可变剪接产生的 mRNA 的质量差异，这可能会完全改变基因功能。在该 R21 应用中，新型 Affymetrix 微阵列能够询问几乎所有已知和预测的外显子，将用于研究红细胞生成过程中选择性剪接的程序化变化。使用阵列原型研究版本的初步合作实验证实，它们可以有效识别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特定阶段变化；例如，以前所未有的分子分辨率促进红细胞转录组的检查。由不同分化阶段的培养的人类和小鼠红系祖细胞 RNA 制备的探针将与微阵列杂交并进行分析，以阐明外显子表达模式的变化。提出了两个目标：（1）对构成人类和小鼠模型中正常红系选择性剪接程序的前 mRNA 剪接中的阶段特异性开关进行全面分析，以及（2）通过测量响应于 RNAi 介导的剪接因子表达水平敲低而发生的剪接变化，从功能上评估所选剪接因子蛋白在红系分化中的作用。这些目标的成功实现将为红细胞选择性剪接计划的范围提供重要的新见解；促进未来拼接开关机制的机理研究；并刺激红系细胞质、细胞骨架和膜蛋白的新型亚型的功能分析。剪接调节中的遗传干扰已在包括肌肉和大脑在内的其他组织中得到描述。目前的研究可能为发现红细胞中新型剪接缺陷奠定基础。最终，模型系统中正在开发的用于治疗性纠正剪接缺陷的方法也可能允许纠正红细胞中的此类缺陷。