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

摘要/项目摘要 虽然经常作为不同的实体进行研究，但转录和RNA加工在 真核生物。我们之前在发芽和分裂酵母中的结果表明，剪接体可以迅速和 一旦POL II合成了内含子，就可以有效地将其移除。然而，有时拼接完全是 被压制，使成绩单成为“死胡同”。这些未拼接的新生转录本不能通过POLIA 卵裂，并被降解。我们假设“生产性”(拼接和 多腺苷)和“末端”转录决定了哺乳动物细胞的基因表达。此外，这一点 机制在应激条件下可能是至关重要的，因为转录通读是一种常见的特征 由感染、癌症、渗透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，他们正在对 来自NHLBI的RFA关于为细胞提供弹性的正常生物学机制。纽格鲍尔博士是一位 他是一名生物化学家，擅长转录和剪接，而皮莱博士是一名造血生物学家， 在红血球种群的产生和特征方面的专门知识。我们的提案调查了 在红细胞生成(红细胞生成或EP)过程中转录和RNA处理之间的耦合。 成熟去核红细胞从未成熟的造血祖细胞经过高度的 由众多外源信号引导的调控分化程序。这种区别的特点是 通过转录组的戏剧性变化，产生一个成熟的红细胞，本质上是一个血红蛋白工厂。 B-珠蛋白是成熟红系细胞中含量最丰富的转录本，已成为先驱研究的关键模型。 前信使核糖核酸剪接和信使核糖核酸稳定性的研究。我们假设积极的和消极的反馈之间 剪接和转录是红系成熟的重要决定因素，它必须对 导致组织缺氧的生理条件(如怀孕、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实验室开发的两个定制的新生RNA-Seq策略：Single 分子内含子跟踪(SMIT)和新生RNA的长阅读测序。在目标1中，我们将利用体外培养的 原代CD34+细胞与人红系分化的培养模型 促红细胞生成素和其他营养因子生成红系细胞，并验证上述假设。目标2将 探索共转录RNA处理如何有助于应激EP期间的转录变化。这 因此，这项研究开创了实验系统的先河，使我们能够精确定位 依靠转录和剪接来维持细胞内环境的稳定。

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.