Erythroid stage-specific transcriptome expression, dynamics, and regulation

描述（由申请人提供）：主要关注晚期红系转录组的全球特征，重点是检测新的转录本和已知转录本的新亚型，转录组在晚期红细胞生成过程中的动态进化，以及进化上保守的替代剪接程序形成转录组的机制。分化的红母细胞执行多样化和动态的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定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控与人类健康极其相关，因为错误调控是许多人类疾病的主要原因，但红细胞剪接程序、其调控及其在红细胞生物学中的重要性仍然知之甚少。本项目提出通过RNA-Seq分析和先进的生物信息学策略对红细胞阶段特异性转录组进行全球分析，以解决这些问题，并为许多其他研究红细胞分化和红细胞生物学的研究者提供丰富的新信息。为了探索保守的哺乳动物红系选择性剪接程序调节红系关键功能的假设，在红系分化、选择性剪接调节和深度测序数据计算分析方面具有专业知识的研究人员共同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分类的红细胞（原红细胞以及嗜碱性、多色和正色红细胞）定义小鼠红细胞阶段特异性转录组。RNA-seq数据的先进计算分析可以比较分化阶段之间以及红系和非红系细胞之间的差异，以表征红系异构体多样性和选择性剪接中的阶段特异性开关，这些开关暗示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细胞特异性和异构体表达的动态开关，这些开关表明红细胞的关键功能，除了突出小鼠和人类细胞之间存在的异构体差异。目标3提出对目标1和目标2中定义的保守的选择性剪接事件进行机制研究，使用计算和生化方法分析顺式调控序列和剪接因子蛋白，指导这些剪接网络。最终，这项工作应该揭示剪接调节网络(s)，协调程序剪接在分化红系细胞。预期这项工作的长期效益包括通过替代蛋白同种异构体大大提高了对红细胞生物过程调节的认识。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。

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.