Regulation of R-loop Formation and Genome Stability by ADAR1

ADAR1 对 R 环形成和基因组稳定性的调节

• 批准号: 10004042
• 负责人: KAZUKO NISHIKURA
• 金额: $ 47.95万
• 依托单位: WISTAR INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10004042
• 关键词: ADAR1; Adenosine; Affect; Affinity Chromatography; Amyotrophic Lateral Sclerosis; Apoptosis; Apoptotic; Apraxias; Ataxia; Autoimmune Diseases; Bees; Biological; Biological Process; Cells; Cellular Stress; Centromere; Chimeric Proteins; Chromosomes; Complex; DNA; DNA Damage; DRADA2b protein; Development; Disease; Dissociation; Double-Stranded RNA; Elements; Embryo; Evolution; Family member; Fragile X Syndrome; Frontotemporal Dementia; Gene Family; Genes; Genetic Transcription; Genome Stability; Genomic Instability; Goals; Grant; Hela Cells; Human Genome; Hybrids; In Vitro; Inflammatory Response; Inosine; Interferons; Knockout Mice; Knowledge; Laboratories; Location; Maintenance; Mediating; MicroRNAs; Mitogen-Activated Protein Kinases; Mitotic; Molecular; Monoclonal Antibodies; Mutation; Natural Immunity; Nucleic Acids; Oligonucleotides; Pathogenesis; Patients; Play; Production; Proteins; RNA; RNA Degradation; RNA Editing; RNA Interference; RNA helicase A; RNA-specific adenosine deaminase 3; Recombinant Proteins; Regulation; Repetitive Sequence; Research; Resolution; Retrotransposon; Ribonucleases; Role; Short Interspersed Nucleotide Elements; Structure; Subgroup; System; TERF1 gene; Testing; Time; Transcript; adenosine deaminase; biological adaptation to stress; design; embryo cell; experimental study; human disease; in vivo; knock-down; mRNA Decay; member; mutant; pre-miRNA; pri-miRNA; pseudotoxoplasmosis syndrome; reconstitution; replication stress; response; ribonuclease H1; telomere; vertebrate genome

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.

项目摘要 阿达尔（作用于RNA的腺苷脱氨酶）将腺苷残基转化为肌苷（A-to-IRNA 编辑）在双链RNA中。在过去27年的拨款之初，我们确定了 ADAR 1是阿达尔基因家族的第一个成员。这反过来又导致了ADAR 2和ADAR 3的鉴定。 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 重点是了解ADAR 1的生物学功能。ADAR 1似乎有多种功能， 编辑依赖性和其他编辑无关性，在选择基因的蛋白质编码中， 逆转录转座子衍生的重复元件，先天免疫的抑制，RNA干扰的调节，以及 应激反应即便如此，目前尚不清楚这些已经描述的ADAR 1功能是否是 ADAR 1基因在脊椎动物基因组进化过程中得以保留的原因。 新生RNA通常在转录后与其模板DNA链解离，但偶尔也会发生这种情况。 新转录的RNA形成稳定的RNA：DNA杂交体，其结果之一是留下有义DNA 以单链形式存在。这种结构被称为R环，并导致转录失败和转录不稳定。 基因组，导致DNA损伤，突变和复制压力。R环积累导致人类 Aicardi-Goutières综合征（AGS）是一种由炎症引起的严重自身免疫性疾病， 对核酸的反应。有趣的是，在AGS患者的亚组（AGS 6）中，疾病是以下因素的结果： ADAR 1突变实验上，我们已经获得了初步的结果表明，ADAR 1 敲低导致R环的显著积累和有丝分裂灾难。 在下一个资助期内，我们将探索ADAR 1的R环调节功能及其在细胞内的表达。 与维持人类基因组稳定性的机制相关。我们将首先调查R- 使用重组蛋白和重组的R环结构进行体外环解离机制 合成RNA/DNA寡核苷酸。我们将研究如何影响R环解离的效率 通过ADAR 1介导的A到I RNA编辑。我们将在全球范围内确定R环的精确位置 通过分离的R环的RNA和DNA链的DRIP-seq由ADAR 1特异性调节。我们 将可视化特定的染色体区域，如着丝粒和端粒，其中R- 环可以通过ADAR 1使用与区域特异性标记融合的荧光蛋白进行特异性调节， 如CENPA和TRF 1。最后，我们将检验我们的假设，即积累的R环是致病核酸， 在ADAR 1缺失小鼠胚胎中检测到的异常IFN产生和炎症反应的酸， AGS 6患者。从ADAR 1缺失小鼠胚胎和携带ADAR 1的HeLa细胞中分离的R环 AGS 6的突变将通过DRIP-seq进行研究。总之，这些实验将揭示 ADAR 1最重要的体内功能，即维持基因组稳定性。