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

使用单分子 RNA-seq 分析红细胞生成过程中的转录和剪接协调

• 批准号: 10210330
• 负责人: Karla M Neugebauer
• 金额: $ 20.84万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10210330
• 关键词: Acute Erythroblastic Leukemia; Address; Aging; Alternative Splicing; Altitude; Anemia; Biological; Biological Models; Bone Marrow; CD34 gene; Cell division; Cells; Cellular Stress; Characteristics; Chromatin; Coupled; Coupling; Cultured Cells; Custom; DNA Polymerase II; Data; Development; Disease; ENG gene; Equilibrium; Erythroblasts; Erythrocytes; Erythroid; Erythroid Cells; Erythropoiesis; Erythropoietin; Eukaryota; Eukaryotic Cell; Feedback; Fission Yeast; Future; Gene Expression; Gene Expression Regulation; Generations; Genes; Genetic Transcription; Goals; Health; Heat-Shock Response; Hematopoietic; Hematopoietic stem cells; Hemoglobin; Hemorrhage; Homeostasis; Human; Hypoxia; In Vitro; Infection; Introns; Knowledge; Lead; Link; Malignant Neoplasms; Mammalian Cell; Messenger RNA; Methods; Modeling; Molecular; Molecular Machines; Morphology; Mus; National Heart, Lung, and Blood Institute; Output; Oxidative Stress; Oxygen; Physiological; Play; Poly A; Population; Positioning Attribute; Pregnancy; Production; RNA; RNA Processing; RNA Splicing; Regulation; Regulator Genes; Research Personnel; Role; Saccharomycetales; Seminal; Signal Transduction; Spliceosomes; Stress; System; Testing; Tissues; Transcript; Transcription Elongation; Virus Diseases; Work; beta Globin; cytokine; human model; improved; in vitro Model; interest; life history; mRNA Precursor; mRNA Stability; programs; resilience; response; single molecule; transcription termination; transcriptome; transcriptome sequencing; transcriptomics

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.

摘要/项目摘要 尽管经常被研究为不同的实体，但转录和RNA处理在 真核生物。我们以前在萌芽和裂变酵母上的结果表明，剪接体可以迅速而 Pol II合成它们后，有效地删除了介绍。然而，有时剪接完全是完全 被压制，渲染成绩单“死胡同”。这些未剪接的新生成绩单未能经历Polya 乳沟并退化。我们假设“生产”（拼接和 聚腺苷酸化）和“死末端”转录本决定哺乳动物细胞中的基因表达。此外，这个 在压力条件下，机制可能至关重要，因为转录读取是经常特征 由感染，癌症，渗透和氧化应激以及其他疾病引起的细胞应激。 该申请汇集了两个正在回应一个调查人员的完整专业知识 NHLBI的RFA对正常的生物学机制，可为细胞提供弹性。 Neugebauer博士是 具有转录和剪接专业知识的生物化学家，而Pillai博士是造血生物学家 促红色种群及其特征方面的专业知识。我们的建议调查了 红细胞（红细胞生成或EP）生产过程中转录和RNA加工之间的耦合。 成熟的红细胞在经历了高度的高度造血祖细胞中出现 受众多外源信号指导的调节分化程序。这种差异是特征的 通过转录组的戏剧性变化，导致成熟的红细胞本质上是血红蛋白工厂。 B-珠蛋白是成熟红色细胞中最丰富的转录本，已成为开创性的关键模型 研究前MRNA剪接和mRNA稳定性的研究。我们假设在 剪接和转录是红斑成熟的重要决定剂，必须弹性 引起组织缺氧的生理状况（例如怀孕，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，我们建议使用红细胞生成作为模型研究共转录剪接动力学 系统。我们将实施Neugebauer实验室中制定的两种自定义新生RNA-Seq策略：单人 分子内含子跟踪（SMIT）和新生RNA的长读测序。在AIM 1中，我们将使用体外 人类红细胞生成分化的培养模型，其中原代CD34+细胞与 促红细胞生成素和其他营养因子产生红细胞细胞并检验上述假设。 AIM 2意志 探索共转录RNA处理如何在压力EP期间有助于转录组变化。这 从而研究先驱者实验系统，这将使我们能够指出基因调节机制 依靠转录和剪接来维持细胞稳态。

项目成果

Identification of Alternative Polyadenylation in Cyanidioschyzon merolae Through Long-Read Sequencing of mRNA.

• DOI: 10.3389/fgene.2021.818697
• 发表时间: 2021
• 期刊: Frontiers in genetics
• 影响因子: 3.7
• 作者: Schärfen L;Zigackova D;Reimer KA;Stark MR;Slat VA;Francoeur NJ;Wells ML;Zhou L;Blackshear PJ;Neugebauer KM;Rader SD
• 通讯作者: Rader SD

Preparation of Mammalian Nascent RNA for Long Read Sequencing.

• DOI: 10.1002/cpmb.128
• 发表时间: 2020-12
• 期刊: Current protocols in molecular biology
• 作者: Reimer KA;Neugebauer KM
• 通讯作者: Neugebauer KM