Regulation of R-loop Formation and Genome Stability by ADAR1

项目总结 ADAR(作用于RNA的腺苷脱氨酶)将腺苷残基转化为肌苷(A-to-I RNA 编辑)在双链RNA中。在这笔赠款的前27年一开始，我们就确定了 ADAR1，ADAR基因家族的第一个成员。这反过来又导致了ADAR2和ADAR3的识别。 从那时起，我们为A-to-I RNA编辑领域的发展做出了重大贡献，特别是通过 重点了解ADAR1的生物学功能。ADAR1似乎有多种功能，有些 编辑依赖和其他编辑独立，在选择基因的蛋白质重新编码中，编辑 反转录转座子衍生的重复元件，先天性免疫的抑制，RNA干扰的调节，以及 压力反应。即便如此，目前还不清楚这些已经描述的ADAR1函数是否是 为什么ADAR1基因在脊椎动物基因组的进化过程中被保留。 新生的RNA通常在转录后从模板DNA链上解离，但偶尔 新转录的RNA形成了稳定的RNA：DNA杂交体，其结果之一是离开了正义DNA 以单链形式存在。这种结构被称为R-环，并导致转录失败和不稳定的 基因组，导致DNA损伤、突变和复制压力。R-环积累导致人类 艾卡迪-古蒂埃综合征(AGS)等疾病，这是一种由炎症性疾病引起的严重自身免疫性疾病 对核酸的反应。有趣的是，在AGS患者的一个亚组(AGS6)中，这种疾病是由 ADAR1基因突变。实验上，我们已经获得了初步结果，表明ADAR1 基因敲除会导致R-环的显著积累和有丝分裂灾难。 在下一个赠款支持期间，我们将探索ADAR1及其 与维持人类基因组稳定的机制有关。我们将首先调查R- 利用重组蛋白和重组R-环结构进行体外环解离机理的研究 用合成的RNA/DNA寡核苷酸。我们将研究R环解离的效率是如何受到影响的 通过ADAR1介导的A-to-I RNA编辑。我们将在全球范围内确定R环的准确位置 由ADAR1通过滴定序列对分离的R-环的RNA和DNA链进行调节。我们 将可视化特定的染色体区域，如着丝粒和端粒，其中R- 环可能由ADAR1使用融合到区域特异性标记的荧光蛋白来特异性地调节，例如 作为CENPA和TRF1。最后，我们将检验我们的假设，即累积的R环是原因核 在ADAR1缺失的小鼠胚胎中检测到异常干扰素产生和炎症反应的酸 AGS6例。从ADAR1缺失的小鼠胚胎和携带ADAR1的HeLa细胞中分离出R环 AGS6的突变将用Drop-Seq方法进行研究。总而言之，这些实验将揭示进化的 ADAR1在体内最重要的功能，即维持基因组的稳定性。

PROJECT SUMMARY ADAR (adenosine deaminase acting on RNA) converts adenosine residues to inosine (A-to-I RNA editing) in double-stranded RNA. At the very beginning of the previous 27 years of this grant, we identified ADAR1, the first member of the ADAR gene family. This in turn led to the identification of ADAR2 and ADAR3. Since then we have made major contributions to development of the A-to-I RNA editing field, in particular by focusing on understanding the biological functions of ADAR1. ADAR1 seems to have multiple functions, some editing-dependent and the others editing-independent, in protein-recoding of select genes, editing of retrotransposon derived repeat elements, suppression of innate immunity, regulation of RNA interference, and stress response. Even so, it is not yet clear whether these already described ADAR1 functions are the reasons why the ADAR1 gene has been retained over the course of evolution of the vertebrate genome. Nascent RNA usually dissociates from its template DNA strand after transcription, but occasionally the newly transcribed RNA forms a stable RNA:DNA hybrid, one consequence of which is leaving the sense DNA in a single-stranded form. This structure is called an R-loop, and causes abortive transcription and instability of the genome, resulting in DNA damage, mutations, and replication stress. R-loop accumulation leads to human diseases such as Aicardi-Goutières syndrome (AGS), a severe autoimmune disease caused by inflammatory responses to nucleic acids. Interestingly, in a subgroup of AGS patients (AGS6), the disease is a result of mutations in ADAR1. Experimentally, we have obtained preliminary results suggesting that ADAR1 knockdown results in significant accumulation of R-loops and mitotic catastrophe. During the next grant support period, we will explore the R-loop regulatory function of ADAR1 and its relevance to the mechanisms that maintain the stability of the human genome. We will first investigate the R- loop dissociation mechanism in vitro using recombinant proteins and a reconstituted R-loop structure made with synthetic RNA/DNA oligonucleotides. We will examine how the efficiency of R-loop dissociation is affected by A-to-I RNA editing mediated by ADAR1. We will determine globally the precise locations of R-loops specifically regulated by ADAR1 by DRIP-seq of both the RNA and the DNA strands of isolated R-loops. We will visualize particular chromosome regions, such as centromeres and telomeres, where persistence of R- loops may be specifically regulated by ADAR1 using fluorescent proteins fused to region specific markers such as CENPA and TRF1. Finally, we will test our hypothesis that accumulated R-loops are the causative nucleic acids for aberrant IFN production and inflammatory responses detected in ADAR1 null mouse embryos and AGS6 patients. R-loops isolated from ADAR1 null mouse embryos and HeLa cells carrying the ADAR1 mutations of AGS6 will be investigated by DRIP-seq. Together, these experiments will reveal the evolutionarily most important in vivo function of ADAR1, namely maintenance of genome stability.