Probing the Function of Long Non-Coding RNAs in Differentiation

探究长非编码 RNA 在分化中的功能

• 批准号: 10653269
• 负责人: DAVID L SPECTOR
• 金额: $ 76.8万
• 依托单位: COLD SPRING HARBOR LABORATORY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-20 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10653269
• 关键词: Biological Models; Candidate Disease Gene; Cell physiology; Disease; Embryo; Embryonic Development; Exhibits; Gene Expression; Genes; Genomics; Goals; Investigation; Knock-out; Mass Spectrum Analysis; Mus; Nuclear; Pathway interactions; Process; Proteins; RNA; Research; Role; Signal Transduction; Untranslated RNA; cell type; chromatin isolation by RNA purification sequencing; embryonic stem cell; human disease; insight; mouse development; overexpression; pluripotency; programs; stem cell differentiation; transcriptome; transcriptome sequencing; treatment strategy

PROJECT SUMMARY The over-arching focus of my research program has been to identify spatial and temporal aspects of regulating gene expression. Our recent emphasis has incorporated mouse embryonic stem cells (ESCs) as a model system to understand gene dynamics and various aspects of gene expression, including the role of long non- coding RNAs (lncRNAs). LncRNAs represent an exciting class of tens of thousands of RNAs among which very few have thus far been functionally characterized. A number of lncRNAs have been shown to exhibit cell- type specific expression, localization to subcellular compartments, and association with human diseases suggesting that they have critical roles in many cellular processes. The long-term goal of this project is to identify the mechanism of action of several lncRNAs identified in an RNA-seq screen that have roles in pluripotency and/or differentiation, and potentially represent different functional classes, Such analysis will provide insight into how these lncRNAs regulate differentiation through their impacts on gene expression and/or nuclear organization. Here, we propose to characterize an exciting nuclear-retained lncRNA, Platr4, that is highly expressed in pluripotent ESCs and is significantly down-regulated upon ESC differentiation. We will examine the impact of Platr4 knockout and over-expression on global gene expression in ESCs. In addition, Platr4 expression will be examined throughout embryonic development and the impact of Platr4 knockout will be examined in developing mouse embryos to identify critical points of function and candidate genes and pathways regulated by Platr4 in early mouse development. Based upon preliminary studies, the role of Platr4 in mesodermal and endodermal lineage commitment will be an immediate focus of investigation. The functional interactions of Platr4 will be identified by an RNA-tagging strategy combined with RNA-seq (ChIRP-seq) to identify genomic interactions of Platr4, and with mass spectrometry (ChIRP-MS) in order to identify proteins interacting with Platr4. Together, the proposed studies will provide important insights into the functional role of Platr4 in lineage commitment and differentiation, and will provide new insights as to how regulatory signals instructed by a long non-coding RNA can impact differentiation and potentially reprogramming.

项目摘要 我的研究计划的重点是确定调节的空间和时间方面 基因表达。我们最近的重点是将小鼠胚胎干细胞（ESC）作为模型 系统，以了解基因动态和基因表达的各个方面，包括长非 编码RNA（lncRNA）。lncRNA代表了一个令人兴奋的类别，其中有数万种RNA， 到目前为止，很少有功能特征。许多lncRNA已经显示出细胞- 型特异性表达、亚细胞区室定位及与人类疾病的关系 这表明它们在许多细胞过程中具有关键作用。该项目的长期目标是 确定在RNA-seq筛选中鉴定的几种lncRNA的作用机制， 多能性和/或分化，并可能代表不同的功能类别。 深入了解这些lncRNA如何通过影响基因表达来调节分化 核组织。在这里，我们提出了一个令人兴奋的核保留lncRNA，Platr 4， 在多能ESC中高度表达，并在ESC分化后显著下调。我们将 研究Platr 4敲除和过表达对ESC中整体基因表达的影响。此外，本发明还提供了一种方法， Platr 4表达将在整个胚胎发育过程中进行检测，Platr 4敲除的影响将在胚胎发育过程中进行。 在小鼠胚胎发育中进行检查，以确定功能和候选基因的关键点， Platr 4在小鼠早期发育中调节的途径。根据初步研究，Platr 4的作用 在中胚层和内胚层谱系中的定型将是研究的直接焦点。功能 Platr 4的相互作用将通过与RNA-seq（ChIRP-seq）组合的RNA标记策略来鉴定， 鉴定Platr 4的基因组相互作用，并使用质谱法（ChIRP-MS）鉴定蛋白质 与Platr 4互动。总之，拟议的研究将提供重要的见解的功能作用， Platr 4在谱系承诺和分化中的作用，并将提供关于调控信号如何 由长的非编码RNA指导的基因突变可以影响分化和潜在的重编程。