Programmed Changes in Alternative Splicing Within Erythr

Erythr 内选择性剪接的程序化变化

• 批准号: 7087238
• 负责人: JOHN G CONBOY
• 金额: $ 21.28万
• 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 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7087238
• 关键词: RNA interference; RNA splicing; animal genetic material tag; biological models; blood proteins; cell differentiation; cell growth regulation; cell line; clinical research; complementary DNA; developmental genetics; erythroid stem cell; erythropoiesis; gene expression; human genetic material tag; human tissue; laboratory mouse; messenger RNA; microarray technology; molecular probes; nucleic acid hybridization; nucleic acid quantitation /detection; protein quantitation /detection; transfection

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）：在此R21提案中启动的研究的主要远距离目的是表征红细胞替代剪接程序，包括（a）在替代性mRNA剪接中鉴定erythroid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。假设替代剪接程序在调节关键红细胞蛋白的结构中起主要作用，因此这些研究可能刺激新型红细胞蛋白同工型的功能研究。这些概念的原理证明是通过先前对蛋白质4.1R基因中替代前MRNA剪接的阶段特异性转换的研究提供的，这对于红细胞膜机械性能至关重要。先前对红细胞基因表达的全基因组分析已为每个基因的表达水平提供了有价值的定量数据。但是，定量每个基因的mRNA输出的现有技术无法区分通过替代剪接产生的mRNA的定性差异，这可能会完全改变基因功能。在此R21应用中，具有询问几乎所有已知和预测外显子的能力的新型Affymetrix微阵列将用于研究红细胞生成期间替代剪接的程序更改。使用原型阵列的原型研究版本的初步协作实验证实，它们有效地识别了分化细胞类型之间的剪接差异。通过延伸，他们还应检测到单个谱系分化过程中剪接的特异性变化。例如，以前所未有的分子分辨率来促进对红细胞转录组的检查。由分化的不同阶段的培养的人和小鼠红细胞祖细胞RNA制备的探针将与微阵列杂交，并分析以阐明外显子表达模式的变化。提出了两个目的：（1）对阶段特异性开关进行跨MRNA剪接中的特异性开关进行全面分析，该开关构成了人类和小鼠模型中正常的红细胞替代剪接程序，（2）在功能上评估所选剪接因子蛋白在通过测量响应RNAi介导的剪接因子表达水平的敲低而发生的剪接变化来分化。成功实现这些目标应该为红斑替代剪接计划的范围提供重大新见解；促进剪接开关机制的未来机械研究；并刺激细胞质，细胞骨架和膜蛋白的新型同工型的功能分析。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。目前的研究可能为在红细胞细胞中发现新的剪接缺陷提供基础。最终，在模型系统中开发的剪接缺陷治疗校正方法可能还可以纠正红细胞细胞中的此类缺陷。