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: 8728222
• 负责人: JOHN G CONBOY
• 金额: $ 45.24万
• 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/8728222
• 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 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; deep sequencing; erythroid differentiation; expectation; genetic regulatory protein; human disease; improved; innovation; insight; iron metabolism; mRNA Precursor; novel; programs; response; tool; transcriptome sequencing

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.

描述(申请人提供)：主要关注晚期红系转录组(S)的全球特征，重点是检测新的转录本和已知转录组的新的异构体，转录组在红系晚期的动态进化，以及进化保守的选择性剪接程序塑造转录组的机制。分化的红细胞执行不同的和动态的Pre-mRNA选择性剪接程序，该程序与转录程序合作，以确保在细胞获得特殊功能特性时合成适当的阶段特异性蛋白质组。对选择性剪接的正确调控与人类健康密切相关，因为错误的调控是许多人类疾病的主要诱因，然而红系剪接程序、其调控及其在红系生物学中的重要性仍然知之甚少。该项目建议通过RNA-Seq分析对特定阶段的红系转录组进行全球分析，并提出先进的生物信息学策略来解决这些问题，并产生丰富的新信息，供许多其他研究红系分化和红系生物学的研究人员使用。为了探索保守的哺乳动物红系选择性剪接程序调节关键红系功能的假设，在红系分化、选择性剪接调节和深度测序数据的计算分析方面具有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利用高纯度的FACS分选的小鼠骨髓红系细胞(前红系细胞以及嗜碱性、多染和正染红系细胞)确定小鼠红系阶段特异性转录组。对RNA-SEQ数据的高级计算分析能够在分化阶段之间以及红系和非红系细胞之间进行比较，以表征红系异型多样性和替代剪接中的阶段特定开关，这意味着编码蛋白质组的功能变化。AIM 2将对通过FACS分选高度纯化的人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系特异性和同型表达的动态开关，这表明了关键的红系功能，此外还强调了存在于小鼠和人类细胞之间的同型差异。目标3通过使用计算和生化方法分析顺式调控序列和指导这些剪接网络的剪接因子蛋白，对AIMS 1和2中定义的保守的选择性剪接事件进行了机制研究。最终，这项工作应该揭示剪接调控网络(S)，它协调程序性剪接分化红系细胞。这项工作预期的长期益处包括极大地改善对通过替代蛋白亚型调节红系细胞的生物过程的洞察。此外，RNA-Seq数据本身可能会刺激对该转录组的转录和转录后调控的研究。