Elucidating the mechanism by which ADAR1 prevents autoimmunity against self RNA

阐明 ADAR1 预防针对自身 RNA 的自身免疫的机制

• 批准号: 10667182
• 负责人: Charles M Rice
• 金额: $ 25.43万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-08 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10667182
• 关键词: Address; Adenosine; Apoptotic; Autoimmune Diseases; Autoimmunity; Binding; Biological Process; Biology; Bite; Calibration; Cell Death; Cell Death Induction; Cell Differentiation process; Cell Line; Cell Survival; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Co-Immunoprecipitations; Double-Stranded RNA; Enzymes; Face; Foundations; Future; Gene Expression; Genes; Genetic Transcription; Genome; Goals; Grant; Human; Immune response; Immune system; Infection; Innate Immune Response; Inosine; Interferon Type I; Interferons; Introns; Investigation; Knock-out; Knowledge; Libraries; Link; Malignant Neoplasms; Medical; Messenger RNA; Modeling; Modification; Molecular; Mutation; Names; Nucleic Acids; Pathway interactions; Pattern; Pattern recognition receptor; Predisposition; Production; Protein Family; Proteins; RNA; RNA Editing; RNA-Binding Proteins; Reporting; Research; Retroelements; Role; Small Interfering RNA; Structure; Testing; Tissues; Transcript; Translational Repression; Untranslated RNA; Untranslated Regions; Up-Regulation; Viral; Virus; Work; autoinflammatory diseases; base; cancer cell; cancer immunotherapy; cancer therapy; crosslinking and immunoprecipitation sequencing; dsRNA adenosine deaminase; genome-wide; human pluripotent stem cell; insight; knock-down; melanoma; neoplastic cell; novel; pathogen; prevent; promoter; protein kinase R; pseudotoxoplasmosis syndrome; receptor; reconstitution; response; viral RNA

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.

项目概要/摘要 为了预防或限制感染，在检测到外来核酸（例如病毒 RNA）后，会产生干扰素 (IFN)，并 引发由数百个 IFN 刺激基因协调的强效先天免疫反应。 尝试区分外来核酸和自身核酸时面临一些挑战，例如细胞。 通常会产生双链 RNA (dsRNA)，可能会被错误地识别为外来物并导致 自身免疫性的例子是 RNA 编辑酶自然发生的突变。 ADAR1（作用于 RNA 1 的腺苷脱氨酶）会引起一种名为 Aicardi-Goutières 的自身炎症性疾病 综合征的特征是在没有感染的情况下出现异常的干扰素反应。 自身免疫，最近的研究表明，消耗 ADAR1 会使肿瘤细胞对先天或 因此，治疗诱导的免疫反应和 ADAR1 可能是抗癌治疗的一个有希望的靶点。 不幸的是，我们对 ADAR1 如何使细胞将自身与细胞区分开来的基本理解存在差距。 通过这笔资助，我们的目标是解决这一知识差距。 ADAR1 属于将 dsRNA 上的腺苷 (A) 修饰为肌苷 (I) 的蛋白质家族。 to-I 修饰通过未知机制阻止受体蛋白对 dsRNA 的感知 包括 PKR（蛋白激酶 R），它通常会引发翻译抑制和细胞死亡。 推测这些修饰改变了 dsRNA 的二级结构并阻止它们激活 然而，ADAR1 经常以未配对的腺苷碱基为目标，因此 A 到 I 的转换不太可能。 在这里，我们提出了一个新的假设，即一个或多个未知的。 RNA 结合蛋白 (RBP) 参与区分 ADAR1 编辑的 dsRNA 和未编辑的 dsRNA。 优先与含有肌苷的 RNA 结合并阻止它们激活 PKR。 我们将结合使用全基因组和高覆盖率 RBP 靶向 CRISPR 敲除筛选 识别蛋白质因子，这些蛋白质因子与 ADAR1 一起防止自身 RNA 触发免疫反应， 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，以优先考虑候选者进行进一步研究。 初步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 ADAR1 和 PKR 的工作将为未来详细的机制研究奠定基础。 总之，我们的建议有可能开辟新的研究途径来理解 我们的研究可能会揭示RNA中肌苷的生物学功能和重要性。 关于细胞如何区分自身 RNA 和非自身 RNA 的机制见解还将提供关键信息。 开发基于 ADAR1 的癌症免疫疗法。