Erythroid stage-specific transcriptome expression, dynamics, and regulation

红系阶段特异性转录组表达、动态和调控

• 批准号: 8258173
• 负责人: JOHN G CONBOY
• 金额: $ 45.14万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-20 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8258173
• 关键词: Address; Affect; Alternative Splicing; Basophilic Erythroblast; Binding; Biochemical; Bioinformatics; Biological Models; Biological Process; Biology; Bone Marrow; Cell division; Cell physiology; Cells; Characteristics; Complex; Computer Analysis; Cultured Cells; Cytoskeleton; Data; Data Analyses; Defect; Detection; Development; Ensure; Erythroblasts; Erythrocytes; Erythroid; Erythroid Cells; Erythropoiesis; Event; Evolution; Exhibits; Exons; Fetal Liver; Foundations; Future; Gene Expression Profile; Genome; Gifts; Goals; Health; Hereditary Disease; Human; Human Biology; Human Genetics; Introns; Joints; Knockout Mice; Knowledge; Lead; Length; Methods; Mining; Modeling; Mus; Myoblasts; Nucleotides; Oligonucleotides; Pathway interactions; Pattern; Physiological; Population; Post-Transcriptional Regulation; Pronormoblasts; Property; Protein Isoforms; Proteins; Proteome; RNA; RNA Processing; RNA Splicing; RNA analysis; Regulation; Regulatory Element; Research; Research Personnel; Shapes; Sorting - Cell Movement; Splicing Regulation Pathway; Staging; Structure; System; Systems Analysis; Testing; Tissues; Transcript; Transcriptional Regulation; Work; base; chromatin modification; comparative genomics; erythroid differentiation; expectation; genetic regulatory protein; human disease; improved; innovation; insight; iron metabolism; mRNA Precursor; novel; programs; response; tool

DESCRIPTION (provided by applicant): Primary focus will be on global characterization of late erythroid transcriptome(s) with emphasis on detection of new transcripts and new isoforms of known transcripts, dynamic evolution of the transcriptome during late erythropoiesis, and mechanisms by which an evolutionarily conserved alternative splicing program shapes the transcriptome. Differentiating erythroblasts execute a diverse and dynamic pre-mRNA alternative splicing program that cooperates with the transcriptional program to ensure synthesis of the appropriate stage-specific proteome as cells acquire specialized functional properties. Proper regulation of alternative splicing is extremely relevant to human health, for misregulation is a major contributor to many human diseases yet the erythroid splicing program, its regulation, and its importance in erythroid biology remain poorly understood. This project proposes a global analysis of the stage-specific erythroid transcriptome by RNA-Seq analysis and advanced bioinformatic strategies to address these issues and to generate a wealth of new information of use to many other investigators studying erythroid differentiation and erythroid biology. To explore the hypothesis that a conserved mammalian erythroid alternative splicing program regulates critical erythroid functions, investigators with expertise in erythroid differentiation, alternative splicing regulation, and computational analysis of deep sequencing data have come together to propose three specific research aims. Aim 1 will define the mouse erythroid stage-specific transcriptome using highly purified FACS-sorted erythroid cells from bone marrow (proerythroblasts as well as basophilic, polychromatic, and orthochromatic erythroblast stages). Advanced computational analysis of RNA-seq data enables comparison among the differentiation stages and between erythroid and non-erythroid cells, to characterize erythroid isoform diversity and stage-specific switches in alternative splicing that imply functional changes in the encoded proteome. Aim 2 will perform a similar analysis of human erythroblasts that are highly purified by FACS sorting. Comparison of human and mouse data will facilitate the definition of evolutionarily conserved erythroid-specific and dynamic switches in isoform expression that suggests critical erythroid functions, in addition to highlighting isoform differences that exist between mouse and human cells. Aim 3 proposes a mechanistic study of the conserved alternative splicing events defined in Aims 1 and 2 by using computational and biochemical approaches to analyze cis- regulatory sequences and splicing factor proteins that direct these splicing networks. Ultimately, this work should reveal the splicing regulatory network(s) that orchestrate programmed splicing in differentiating erythroid cells. Long term benefits anticipated from this work include greatly improved insights into regulation of biological processes in erythroid cells by alternative protein isoforms. Moreover, the RNA-Seq data itself may stimulate studies of the transcriptional and post-transcriptional regulation of this transcriptome. PUBLIC HEALTH RELEVANCE: Understanding how tissue-specific alternative splicing is regulated is extremely relevant to human health, because splicing mis-regulation is the underlying cause of a significant number of human genetic diseases. This project will lay the foundations for analysis of alternative splicing networks in that operate during differentiation of erythroblasts into mature red cells. This knowledge will stimulate mechanistic studies of normal red cell function and may contribute to understanding of aberrant red cells due to splicing defects.

描述（由申请人提供）：主要关注晚期红细胞转录组的整体表征，重点是检测新转录本和已知转录本的新亚型、晚期红细胞生成期间转录组的动态演变以及进化上保守的选择性剪接程序塑造转录组的机制。分化的成红细胞执行多样化和动态的前mRNA选择性剪接程序，该程序与转录程序合作，以确保当细胞获得专门的功能特性时合成适当的阶段特异性蛋白质组。选择性剪接的适当调节与人类健康极其相关，因为调节不当是许多人类疾病的主要原因，但红细胞剪接程序、其调节及其在红细胞生物学中的重要性仍然知之甚少。该项目提出了通过RNA-Seq分析和先进的生物信息学策略对阶段特异性红细胞转录组进行全球分析，以解决这些问题，并为研究红细胞分化和红细胞生物学的许多其他研究人员提供丰富的新信息。为了探索保守的哺乳动物红细胞选择性剪接程序调节关键红细胞功能的假设，具有红细胞分化，选择性剪接调节和深度测序数据计算分析专业知识的研究人员聚集在一起，提出了三个具体的研究目标。目的1将使用来自骨髓的高度纯化的FACS分选的红系细胞（原成红细胞以及嗜碱性、多色和正染成红细胞阶段）来定义小鼠红系阶段特异性转录组。RNA-seq数据的高级计算分析使得能够在分化阶段之间以及红细胞和非红细胞之间进行比较，以表征红细胞同种型多样性和可变剪接中的阶段特异性开关，这意味着编码蛋白质组的功能变化。目标2将对通过FACS分选高度纯化的人成红细胞进行类似的分析。人类和小鼠数据的比较将有助于定义进化上保守的红细胞特异性和动态开关的亚型表达，这表明关键的红细胞功能，除了突出小鼠和人类细胞之间存在的亚型差异。目的3提出了通过使用计算和生物化学方法来分析指导这些剪接网络的顺式调节序列和剪接因子蛋白质，对目的1和2中定义的保守可变剪接事件进行机制研究。最终，这项工作应该揭示剪接调控网络，协调程序剪接在分化红细胞。从这项工作预期的长期利益包括大大提高了对红系细胞中的生物过程的调节的替代蛋白质亚型的见解。此外，RNA-Seq数据本身可以刺激对该转录组的转录和转录后调控的研究。 公共卫生关系：了解组织特异性选择性剪接是如何调节的与人类健康极其相关，因为剪接错误调节是大量人类遗传疾病的根本原因。该项目将为分析在成红细胞分化为成熟红细胞过程中起作用的可变剪接网络奠定基础。这些知识将刺激正常红细胞功能的机制研究，并可能有助于理解由于剪接缺陷导致的异常红细胞。