Dicer as a repressor of antiviral response in embryonic stem cells

项目总结 早期胚胎发育是哺乳动物生命周期中最脆弱的阶段。胚胎干细胞(ESCs) 在子宫中有一个专门的任务，细胞的快速增殖的有机发育，但他们可以 受到许多环境因素的不利影响，如感染和炎症。目前，我们知道的很少 关于ESCs是如何应对压力的。干扰素系统是天然抗病毒药物的核心部分。 分化的体细胞中的免疫。我们最近证明了胚胎干细胞不能表达干扰素， 这表明ESCs可能没有基于干扰素的功能性抗病毒机制。这一发现提出了重要的 关于ESCS没有如此关键的防御机制的理由以及ESCS如何处理的问题 感染了病毒。使用基于合成dsRNA的无病毒系统，我们出人意料地发现迪格尔 基因敲除的胚胎干细胞(D-/-ESCs)似乎能够表达干扰素，并对增加的细胞毒高度敏感 DsRNA的效应。同样，B2RNA，一种来自转座元件转录产物的细胞RNA， 显示出与dsRNA引起的这些非常相似的效果。这些发现挑战了我们的观点，即缺乏 干扰素应答是胚胎干细胞所固有的。相反，我们的发现表明，干扰素系统的基本机制 在胚胎干细胞中具有功能，但通常可能被DICER抑制。已知细胞RNA与dsRNA 来自受损细胞、错误处理的RNA和转座元件转录本的结构可以诱导 引起细胞损害的抗病毒反应，类似于病毒感染。我们假设，通过抑制干扰素 作为一种前哨酶来防止过度的抗病毒反应，可能会潜在地 在早期胚胎发育过程中危害胚胎干细胞。使用小鼠胚胎干细胞作为模型系统，我们将检验这一假说 通过：1)使用合成的dsRNA作为病毒RNA模拟物来确定主要信号事件的功能 对D-/-ESCs中的干扰素系统至关重要(目标1)，2)使用B2RNA作为与生理相关的细胞RNA来 研究干扰素系统的激活并确定Dester在限制B2RNA诱导的作用中的作用 细胞毒性(目标2)，以及3)从两个质粒载体中用异位表达的DICER重组D-/-ESCs 和合成的DICER-mRNA。重组的ESCS将用于验证从 D-/-ESCs中的dsRNA和B2RNA。拟议项目的成功完成预计将提供 对ESCs独特的免疫特性的重大洞察，特别是关于基于干扰素的抗病毒 先天免疫由迪格尔精心控制，确保早期正常生长发育 生物体发育的阶段。

PROJECT SUMMARY Early embryogenesis is the most vulnerable stage in the life cycle of mammals. Embryonic stem cells (ESCs) in the uterus have a dedicated task of rapid cell proliferation for the organismal development, but they can be adversely affected by many environmental factors, such as infection and inflammation. Currently, we know little about how ESCs cope with stress conditions. The interferon (IFN) system is the central part of antiviral innate immunity in differentiated somatic cells. We have recently demonstrated that ESCs are unable to express IFN, indicating that ESCs may not have a functional IFN-based antiviral mechanism. This finding raises important questions about the rationale for ESCs to not have such a critical defense mechanism and how do ESCs deal with viral infection. Using a synthetic dsRNA-based virus-free system, we have unexpectedly found that Dicer knockout ESCs (D-/-ESCs) seem to be able to express IFN and are highly sensitive to the increased cytotoxic effects of dsRNA. Similarly, B2RNA, a cellular RNA derived from the transcripts of transposable elements, showed remarkably similar effects to these caused by dsRNA. These findings challenge our view that the lack of IFN response is intrinsic in ESCs. Instead, our finding suggests that the basic machinery of the IFN system is functional in ESCs, but it may normally be repressed by Dicer. It is known that cellular RNA with dsRNA structures, derived from injured cells, misprocessed RNA and transcripts of transposable elements, can elicit antiviral responses that cause cellular damage similar to viral infection. We hypothesize that, by repressing IFN response, Dicer may act as a sentinel enzyme to prevent excessive antiviral responses that could potentially harm ESCs during early embryogenesis. Using mouse ESCs as a model system, we will test this hypothesis by: 1) using synthetic dsRNA as viral RNA mimics to determine the functionality of the major signaling events essential for the IFN system in D-/-ESCs (Aim 1), 2) using B2RNA as a physiologically relevant cellular RNA to investigate the activation of the IFN system and determine the role of Dicer in restricting B2RNA-induced cytotoxicity (Aim 2), and 3) reconstituting D-/-ESCs with ectopically expressed Dicer from both a plasmid vector and from synthetic Dicer-mRNA. The reconstituted ESCs will be used to validate the results obtained from dsRNA and B2RNA in D-/-ESCs. The successful completion of the proposed project is expected to provide significant insight into the unique immune properties of ESCs, specifically on how the IFN-based antiviral innate immunity is delicately controlled by Dicer to ensure normal growth and development at the early stage of organismal development.