Analysis of transcription and splicing coordination during erythropoeisis using single molecule RNA-seq

摘要/项目总结 虽然经常作为不同的实体进行研究，但转录和RNA加工在基因组中错综复杂地联系在一起。 真核生物我们以前在芽殖和分裂酵母中的结果表明，剪接体可以快速地， 一旦Pol II合成内含子，就有效地去除内含子。尽管如此，有时拼接完全 压制，使成绩单“死胡同”。这些未剪接的新生转录物不能进行polyA 分裂并降解。我们假设，“生产性”（拼接和 多腺苷酸化的）和“死端”转录物决定哺乳动物细胞中的基因表达。而且这 在应激条件下，这种机制可能至关重要，因为转录通读是一种常见的特征 感染、癌症、渗透压和氧化应激以及其他条件引起的细胞应激。 该应用程序汇集了两名调查人员的互补专业知识，他们正在应对一个 来自NHLBI的RFA对提供细胞弹性的正常生物机制的影响。诺伊格鲍尔博士是一位 生物化学家在转录和剪接的专业知识，而Pillai博士是一个造血生物学家， 红细胞群体的产生及其表征的专业知识。我们的建议调查了 在红细胞产生（红细胞生成或EP）期间转录和RNA加工之间的偶联。 成熟的去核红细胞在经历了高度增殖后从未成熟的造血祖细胞中出现。 由许多外源信号引导的受调节的分化程序。这种分化的特点是 通过转录组的巨大变化，产生了一个成熟的红细胞，它本质上是一个血红蛋白工厂。 b-珠蛋白是成熟红系细胞中最丰富的转录物， 研究前mRNA剪接和mRNA稳定性。我们假设，积极和消极的反馈之间， 剪接和转录是红系成熟的重要决定因素，其必须对 导致组织缺氧的生理条件（如妊娠、高海拔）。由此产生的“压力EP” 增加红细胞的产生，以便向组织输送更多的氧气。 因此，我们建议以红细胞生成为模型，研究共转录剪接动力学 系统我们将实施Neugebauer实验室开发的两种定制新生RNA-Seq策略： 新生RNA的分子内含子追踪（SMIT）和长读段测序。在目标1中，我们将利用体外 人红细胞生成分化的培养模型，其中原代CD 34+细胞与 促红细胞生成素和其它营养因子以产生红细胞并测试上述假设。目标2将 探索如何共转录RNA加工可能有助于转录组的变化，在压力EP。这 因此，这项研究开创了实验系统，使我们能够确定基因调控机制， 依靠转录和剪接来维持细胞内稳态。

ABSTRACT/PROJECT SUMMARY Although often studied as distinct entities, transcription and RNA processing are intricately linked in eukaryotes. Our previous results in budding and fission yeasts show that the spliceosome can quickly and efficiently remove introns as soon as Pol II synthesizes them. Nevertheless, sometimes splicing is completely suppressed, rendering transcripts “dead end”. These unspliced nascent transcripts fail to undergo polyA cleavage and are degraded. We hypothesize that the balance between “productive” (spliced and polyadenylated) and “dead-end” transcripts determines gene expression in mammalian cells. Furthermore, this mechanism is likely crucial under conditions of stress, because transcriptional readthrough is a frequent feature of cellular stresses induced by infection, cancer, osmotic and oxidative stress, and other conditions. The application brings together the complementary expertise of two investigators who are responding to an RFA from NHLBI on normal biological mechanisms that provide cells with resilience. Dr. Neugebauer is a biochemist with expertise in transcription and splicing, while Dr. Pillai is a hematopoietic biologist with expertise in generation of erythroid populations and their characterization. Our proposal investigates the coupling between transcription and RNA processing during production of red blood cells (erythropoiesis or EP). Mature enucleated red blood cells emerge from immature hematopoietic progenitors after undergoing a highly regulated differentiation program guided by numerous exogenous signals. This differentiation is characterized by dramatic changes in the transcriptome, resulting in a mature red cell that is essentially a hemoglobin factory. b-globin, the most abundant transcript in mature erythroid cells, has served as a critical model for pioneering studies in pre-mRNA splicing and mRNA stability. We hypothesize that positive and negative feedback between splicing and transcription are important determinants of erythroid maturation, which must be resilient to physiological conditions (e.g. pregnancy, high altitude) that cause tissue hypoxia. The resulting “Stress EP” increases red cell production in order to deliver more oxygen to the tissues. We therefore propose to investigate co-transcriptional splicing dynamics, using erythropoiesis as a model system. We will implement two custom nascent RNA-Seq strategies developed in the Neugebauer lab: Single Molecule Intron Tracking (SMIT) and long read sequencing of nascent RNA. In Aim 1, we will utilize an in vitro culture model of human erythropoietic differentiation in which primary CD34+ cells are cultured with erythropoietin and other trophic factors to generate erythroid cells and test the above hypotheses. Aim 2 will explore how co-transcriptional RNA processing may contribute to transcriptomic changes during stress EP. This study thereby pioneers experimental systems that will allow us to pinpoint gene regulatory mechanisms that rely on transcription and splicing to maintain cellular homeostasis.