Elucidating the mechanism by which ADAR1 prevents autoimmunity against self RNA

项目总结/摘要 为了防止或限制感染，在感测外源核酸（例如，产生病毒RNA）干扰素（IFN）， 激发由数百个IFN刺激基因协调的强有力的先天免疫应答。然而，细胞面对 当试图区分外来和自身核酸时，存在几个挑战。例如，细胞 通常会产生双链RNA（dsRNA），这些RNA可能被错误地识别为外源RNA， 自身免疫这一点可以通过这样一个事实来证明，即在一种称为RNA编辑酶的天然突变中， ADAR 1（腺苷脱氨酶作用于RNA 1）导致称为Aicardi-Goutières的自身炎症性疾病 在没有感染的情况下以干扰素异常反应为特征的综合征。除了此链接， 自身免疫，最近的研究表明，耗尽ADAR 1使肿瘤细胞对先天或 因此，治疗诱导的免疫应答和ADAR 1可能是抗癌治疗的有希望的靶点。 不幸的是，在我们对ADAR 1如何使细胞从自身分化为 非自身RNA。通过这笔赠款，我们的目标是解决这一知识差距。 ADAR 1属于将dsRNA上的腺苷（A）修饰为肌苷（I）的蛋白质家族。这些A- 通过一种未知的机制，to-I修饰阻止了受体蛋白对dsRNA的感知 包括PKR（蛋白激酶R），其通常触发翻译抑制和细胞死亡。已经 推测这些修饰改变了双链RNA的二级结构，并阻止它们激活 PKR。然而，ADAR 1经常靶向未配对的腺苷碱基，因此不太可能将A转化为I 对dsRNA结构造成重大破坏。在这里，我们提出了一个新的假设，一个或多个未知的 RNA结合蛋白（RBP）参与区分ADAR 1编辑的dsRNA与未编辑的dsRNA。该RBP可以 优先与含肌苷的RNA结合并阻止它们激活PKR。 我们将使用全基因组和高覆盖率RBP靶向CRISPR敲除筛选的组合 鉴定与ADAR 1一起阻止自身RNA触发免疫反应的蛋白质因子， 细胞死亡我们将使用半阵列siRNA文库评估潜在的命中，并包括额外的读数。 例如PKR激活和IFN产生，以优先考虑进一步研究的候选者。最后，我们将启动 初步的机制研究，以确定它们的RNA相互作用和潜在的蛋白质相互作用， ADAR 1和PKR。这项工作将为今后详细的机理研究奠定基础。 总之，我们的建议有可能开辟新的研究途径，以了解 RNA内肌苷的生物学功能和重要性。我们的研究可能会揭示新的和范式转变 对细胞如何区分自我与非自我RNA的机械见解。它还将提供关键信息， 开发基于ADAR 1的癌症免疫疗法。

Project Summary/Abstract To prevent or limit infection, upon sensing foreign nucleic acids (e.g., viral RNA) interferon (IFN) is produced and elicits a potent innate immune response orchestrated by hundreds of IFN-stimulated genes. However, cells face several challenges when attempting to differentiate between foreign and self nucleic acids. For instance, cells often produce double-stranded RNAs (dsRNA) that can be mistakenly recognized as foreign and lead to autoimmunity. This is exemplified by the fact that naturally occurring mutations in an RNA-editing enzyme called ADAR1 (adenosine deaminase acting on RNA 1) cause an autoinflammatory disorder named Aicardi-Goutières syndrome characterized by an aberrant IFN response in the absence of infection. In addition to this link to autoimmunity, recent studies have demonstrated that depleting ADAR1 sensitizes tumor cells to innate or therapy-induced immune responses and ADAR1 may therefore be a promising target for anti-cancer therapies. Unfortunately, there is a gap in our basic understanding of how ADAR1 enables cells to differentiate self from non-self RNAs. With this grant, we aim to address this knowledge gap. ADAR1 belongs to a family of proteins that modifies adenosines (A) to inosines (I) on dsRNA. These A- to-I modifications, through an unknown mechanism, prevent the sensing of dsRNAs by receptor proteins including PKR (protein kinase R), which normally triggers translational inhibition and cell death. It has been speculated that these modifications alter the secondary structures of dsRNAs and prevent them from activating PKR. However, ADAR1 frequently targets unpaired adenosine bases, and therefore A-to-I conversion is unlikely to cause major disruption to dsRNA structures. Here, we propose a novel hypothesis that one or more unknown RNA-binding proteins (RBPs) are involved in differentiating ADAR1-edited vs. unedited dsRNAs. This RBP may preferentially bind to inosine-containing RNAs and prevent them from activating PKR. We will use a combination of genome-wide and high-coverage RBP-targeted CRISPR knockout screens to identify protein factors, which together with ADAR1, prevent self RNA from triggering immune responses and cell death. We will evaluate potential hits using a semi-arrayed siRNA library and include additional readouts such as PKR activation and IFN production to prioritize candidates for further investigation. Lastly, we will initiate preliminary mechanistic studies to determine their RNA interactions and potential protein interactions with ADAR1 and PKR. This work will lay the foundation for future detailed mechanistic studies. In conclusion, our proposal has the potential to open new avenues of research to understand the biological functions and importance of inosines within RNA. Our study may reveal novel and paradigm-shifting mechanistic insights into how cells differentiate self vs. non-self RNA. It will also provide critical information for developing ADAR1-based cancer immunotherapies.