Probing the Function of Long Non-Coding RNAs in Differentiation

项目总结 我的研究计划的主要重点一直是确定监管的空间和时间方面 基因表达。我们最近的重点是将小鼠胚胎干细胞(ESCs)作为模型 系统了解基因动力学和基因表达的各个方面，包括长非的作用 编码RNA(LncRNAs)。LncRNA代表了一类令人兴奋的数万种RNA，其中 到目前为止，很少有人被描述为功能上的特征。许多lncRNA已被证明展示了细胞- 类型特异性表达、亚细胞室定位及其与人类疾病的关系 这表明它们在许多细胞过程中起着关键作用。这个项目的长期目标是 确定在RNA-SEQ屏幕中识别的几个在以下方面具有作用的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，这种分析将 洞察这些lncRNAs如何通过影响基因表达来调节分化 和/或核组织。在这里，我们建议描述一种令人兴奋的保留核的lncRNA，Platr4，它 在多能胚胎干细胞中高表达，在胚胎干细胞分化过程中显著下调。我们会 研究Platr4基因敲除和过度表达对ESCs整体基因表达的影响。此外, Platr4的表达将在整个胚胎发育过程中被检测，Platr4基因敲除的影响将 在小鼠胚胎发育过程中进行检测，以确定功能关键点和候选基因 小鼠发育早期受Platr4调控的通路。根据初步研究，Platr4的作用 在中胚层和内胚层谱系中，承诺将是一个直接的研究重点。功能界别 Platr4的相互作用将通过RNA标签策略和RNA-seq(chirp-seq)相结合来识别 鉴定Platr4的基因组相互作用，并用质谱仪(Chirp-MS)鉴定蛋白质 与Platr4交互。综上所述，拟议的研究将为了解脑血管紧张素转换酶的功能作用提供重要见解 Platr4在世系承诺和分化方面的研究，并将提供关于监管信号如何 在长的非编码RNA的指导下，可以影响分化和潜在的重新编程。

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.