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（由申请方提供）：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）鉴定前mRNA选择性剪接中的红细胞阶段特异性开关，和（B）阐明主要剪接调控蛋白在实现该程序中的作用。假设选择性剪接程序在调节关键红细胞蛋白质的结构中起主要作用，因此这些研究可能会刺激新的红细胞蛋白质异构体的功能研究。这些概念的原理的证据是由以前的研究提供的阶段特异性开关的替代前mRNA剪接的蛋白质4.1R基因，这是关键的红细胞膜的机械性能。以前的全基因组分析红细胞基因表达提供了有价值的定量数据，每个基因的表达水平。然而，用于定量来自每个基因的mRNA输出的现有技术不能区分由可变剪接产生的mRNA的定性差异，这可能完全改变基因功能。在这个R21应用中，具有询问几乎所有已知和预测的外显子的能力的新型Affytron微阵列将用于研究红细胞生成过程中可变剪接的程序性变化。使用原型研究版本的阵列进行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，它们还应该检测单个谱系分化期间剪接的阶段特异性变化;例如，有助于以前所未有的分子分辨率检查红细胞转录组。将从处于不同分化阶段的培养的人和小鼠红系祖细胞RNA制备的探针与微阵列杂交并分析以阐明外显子表达模式的变化。提出了两个目标：（1）对前体mRNA剪接中的阶段特异性开关进行全面分析，所述阶段特异性开关构成人和小鼠模型中的正常红细胞选择性剪接程序，和（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，功能性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应提供显着的红细胞选择性剪接程序的范围内的新见解，促进未来的剪接开关机制的机制研究，并刺激红细胞细胞质，细胞骨架和膜蛋白的新亚型的功能分析。在其他组织，包括肌肉和大脑中的剪接调控的遗传紊乱已被描述，本研究可能提供一个基础，发现新的剪接缺陷的红细胞。最终，在模型系统中开发的用于治疗性校正剪接缺陷的方法也可以允许校正红系细胞中的此类缺陷。