RNA editing controls pulmonary endothelial pathophenotypes in pulmonary hypertension

摘要 这项提议的主要目标是产生一个肺血管疾病的科学发现计划 以确保伍德科克博士成长为一名独立研究科学家的稳健道路。这一目标 将通过积极的指导、教学培训、充实活动和 研究。这项研究的核心是RNA编辑的作用，这是一种转录后调节 机制，在肺动脉高压中。肺动脉高压(PH)是一种无法治愈的致命疾病。早些时候 肺动脉内皮细胞(PAECs)的凋亡是PH发生发展的重要触发因素。然而， 关于控制PAEC病理表型的机制或关键因素，存在严重的知识差距 导致了这种致命的疾病，目前还没有针对这种关键细胞类型的有效治疗方法。 作用于RNA 1的腺苷脱氨酶(ADAR1)是一种双链RNA编辑酶，它将 在基因组编码的RNA转录本中，腺苷到肌苷(A-to-I)，并在基因调控和 心脏功能。然而，ADAR1介导的RNA编辑在肺血管调节中的作用 功能都是未知的。我们的初步数据表明，ADAR1在PAEC的调节中起着至关重要的作用 生存和胞浆先天免疫反应。为了确定PAEC中丰富的基因被修饰和 在ADAR1 RNA编辑活性的调控下，我们最近进行了ADAR1基因敲除的体外RNA测序 太平洋经济合作组织。从这一分析中我们发现，昼夜节律基因nocturin(Noct)含有两个活性的adar1 rna。 正在编辑站点。未编辑的NOCT转录本是稳定的，并在缺乏ADAR1 RNA编辑的PAEC内积累。 NOCT强制表达上调关键干扰素转录因子IRF7并促进细胞凋亡 太平洋经济合作组织。NOCT基因敲除(KO)小鼠可减轻缺氧和IL6暴露所致的PH。最后，沉默的夜 恢复ADAR1缺乏所致的PAEC凋亡和免疫反应。在我们的初步研究中，我们 通过展示ADAR1 RNA编辑可以控制noct表达来探索这一概念的关键要素 调节PAEC功能和PH值。根据这些发现，我们假设ADAR1 RNA编辑缺陷 通过异常的noct诱导肺动脉内皮细胞凋亡，从而促进PH的发生发展。 驱使先天免疫激活。这一假设将在以下具体的实验中得到解决 目的：(1)确定noct在先天免疫依赖的PAEC生存反应中的作用；(2) 确定ADAR1特异性RNA编辑对PAEC中noct表达和功能的影响； 明确血管内皮细胞ADAR1缺陷对在体肺组织中NOCT-干扰素信号的影响。这一新机制 可能对RNA编辑如何调节与PH相关的PA内皮功能具有重要意义 发病机制及其阐明将促进我们对PH的发展的理解，并最终导致新的 发展有效的治疗方法来治疗PH的策略。这个项目将为我提供先进的专业知识 在RNA生物学和肺生理学方面，这将加强我成为一名独立研究员的发展。

ABSTRACT The primary objective of this proposal is to generate a scientific discovery program in pulmonary vascular disease to ensure a robust pathway for Dr. Woodcock to develop into an independent research scientist. This objective will be accomplished through a combination of active mentorship, didactic training, enrichment activities, and research. The core of the research focuses on the role of RNA editing, a post-transcriptional regulation mechanism, in pulmonary hypertension. Pulmonary hypertension (PH) is a lethal disease without a cure. Early apoptosis in pulmonary artery endothelial cells (PAECs) is a key trigger for the development of PH. However, there is a critical knowledge gap regarding the mechanisms or key factors controlling PAEC pathophenotypes that drive this deadly disease, and no effective therapeutics exist for PH that target this key cell type. Adenosine deaminase acting on RNA 1 (ADAR1) is a double-stranded RNA editing enzyme that converts adenosine to inosine (A-to-I) in genome-encoded RNA transcripts and plays a vital role in gene regulation and cardiac function. However, the roles of ADAR1-mediated RNA editing in the regulation of pulmonary vascular function are unknown. Our preliminary data indicate that ADAR1 plays a crucial role in the regulation of PAEC survival and the cytosolic innate immunity response. To identify enriched genes in PAECs that are modified and regulated by ADAR1 RNA editing activity, we recently performed in vitro RNA sequencing in ADAR1 knockdown PAECs. From this analysis, we found that the circadian gene nocturnin (NOCT) contains two active ADAR1 RNA editing sites. Non-edited NOCT transcript is stable and accumulates within PAECs lacking ADAR1 RNA editing. Forced expression of NOCT upregulated key interferon transcriptional factor IRF7 and promoted apoptosis in PAECs. NOCT knockout (KO) mice mitigated PH induced by hypoxia and IL6 exposure. Lastly, silenced NOCT restored PAEC apoptosis and immunity response triggered by ADAR1 deficiency. In our pilot studies, we explored key elements of this concept by showing that ADAR1 RNA editing can control NOCT expression to regulate PAEC functions and PH. Given these findings, we hypothesize that ADAR1 RNA editing deficiency promotes PH development due to induction of pulmonary artery endothelial cell apoptosis via aberrant NOCT- driven innate immunity activation. This hypothesis will be addressed in the experiments of the following Specific Aims: (1) to determine the role of NOCT in the innate immunity-dependent PAEC survival response; (2) to determine the effect of ADAR1-specific RNA editing on NOCT expression and function in PAECs; and (3) to define the impact of endothelial ADAR1 deficiency on NOCT-IFN signaling in PH in vivo. This novel mechanism may have important implications for how RNA editing regulates PA endothelial functions related to PH pathogenesis, and its elucidation will advance our understanding of PH development and ultimately lead to novel strategies for developing effective therapeutics to treat PH. This project will provide me with advanced expertise in RNA biology and pulmonary physiology that will strengthen my development into an independent investigator.