Biology of RNA G-quadruplexes

RNA G 四链体的生物学

• 批准号: 10335156
• 负责人: Pavel Ivanov
• 金额: $ 37.59万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10335156
• 关键词: 3-Dimensional; 7-deazaguanine; Affect; Base Pairing; Binding; Binding Proteins; Biochemical; Biological; Biological Assay; Biological Process; Biological Testing; Biology; Biophysics; Cancer Biology; Cell physiology; Cells; Cellular Stress; Collaborations; DNA; Data; Development; Disease; Elements; Etiology; Eukaryotic Cell; Event; Functional disorder; Gene Expression; Genes; Genetic Transcription; Genetic Translation; Goals; Growth; Guanine; Guanosine; Homeostasis; Human; In Vitro; Intervention; Investigation; Ions; Laboratories; Lead; Malignant Neoplasms; Mediating; Messenger RNA; Metabolism; Methods; Molecular; Molecular Target; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Nucleic Acid Regulatory Sequences; Nucleic Acids; Pathologic; Pathology; Pathway interactions; Pharmacology; Physiological; Poly A; Process; Proteins; Proteomics; Publishing; RNA; RNA Binding; RNA Sequences; RNA metabolism; Regulation; Regulatory Element; Reporting; Research; Ribonucleic Acid Regulatory Sequences; Role; Stress; Structure; System; Techniques; Testing; Therapeutic; Transcript; Translations; Work; base; biological adaptation to stress; biophysical techniques; genetic manipulation; genome-wide analysis; human disease; in vivo; interest; mRNA Stability; novel; novel strategies; novel therapeutics; physiologic stressor; prevent; telomere; three dimensional structure; transcriptome; tumorigenic

Summary G-quadruplexes (G4s) are non-canonical secondary structures in nucleic acids that are formed by guanine-rich sequences. G4 structural and functional studies have largely focused on DNA G4s, and the number of biological functions assigned to these motifs has grown rapidly since the discovery of their involvement in telomere biology. RNA G4s (RG4s) are less studied, but interest is increasing due to their association with multiple processes. A comprehensive understanding of how RNA G4s contribute to cell physiology and pathophysiology is the long-term research goal of the applicant. Multiple reports clearly demonstrate that G4s are enriched in mRNA 5’- and 3’-untranslated regulatory regions. There is an increasing evidence that RG4s control gene expression at transcriptional and post- transcriptional levels, although such data is largely based on in vitro studies. Testing the biological significance of RG4s requires proving that RG4s exist in vivo. Intriguingly, RG4s appear predominantly unfolded in eukaryotic cells, whereas they are readily folded in vitro, suggesting that in cells RG4s are constitutively recognized and actively unfolded. We hypothesize that RG4 folding-unfolding regulates mRNA homeostasis. This hypothesis is based on our analysis of human transcriptome that identifies RG4s as stress-responsive RNA elements. We will test this hypothesis with three specific aims. In AIM1, we will determine and characterize the fraction of the human transcriptome that contains putative stress-responsive RG4s in living cells. In AIM2, we will identify bona fide RG4-binding proteins using a novel approach based on proteomic/biochemical analysis of interactions between the 7-deazaguanine RNA derivatives and proposed binding factors. In AIM 3, we will use functional assays to determine the biological significance of RG4s in mRNA stability, localization and translation. We will use biophysical and biochemical methods to validate selected RG4 candidates. This work will elucidate how RG4-mediated functions contribute to cellular mRNA homeostasis, and will identify physiologically significant RG4-binding partners, which in turn may reveal molecular targets and pathways with therapeutic potential. The understanding of cellular functions of RG4 motifs is particularly relevant to the biology of cancer and neurodegeneration. RNA regions containing RG4s significantly overlap with regions containing disease- associated mutations. The proposed studies may elucidate molecular events underlying normal and pathological aspects of cell physiology, and identify events contributing to the tumorigenic or neurodegenerative changes.

摘要 G-四链体(G4S)是核酸中的非正则二级结构，由 富含鸟嘌呤序列。G4的结构和功能研究主要集中在DNA G4S上，而 自从这些基序的发现以来，分配给这些基序的生物学功能的数量迅速增加 参与端粒生物学。对RNAG4S(RG4s)的研究较少，但由于其 与多个进程关联。全面了解RNAG4S对细胞的贡献 生理学和病理生理学是申请者的长期研究目标。 多份报告清楚地表明，G4S富含5‘和3’未翻译的调控基因 地区。越来越多的证据表明，RG4在转录和转录后控制基因表达。 转录水平，尽管这些数据主要基于体外研究。测试其生物学意义 RG4的研究需要证明RG4在体内存在。有趣的是，RG4似乎主要是在 真核细胞，而它们在体外很容易折叠，这表明在细胞中RG4是结构性的 被认可并积极展开。我们假设RG4折叠-去折叠调节mRNA的动态平衡。 这一假设是基于我们对人类转录组的分析，该转录组确定RG4是应激反应的 RNA元素。我们将通过三个具体目标来检验这一假设。在AIM1中，我们将确定和 描述人类转录组中含有假定的应激反应RG4的部分 细胞。在AIM2中，我们将使用一种新的方法鉴定真正的RG4结合蛋白，该方法基于 蛋白质组学/生化分析7-去氮鸟嘌呤RNA衍生物与建议的 约束性因素。在AIM 3中，我们将使用功能分析来确定RG4在 信使核糖核酸的稳定性、定位和翻译。我们将使用生物物理和生化方法来验证 选定的RG4候选人。这项工作将阐明RG4介导的功能如何对细胞mRNA做出贡献 动态平衡，并将识别生理上重要的RG4结合伙伴，这反过来可能揭示 具有治疗潜力的分子靶点和途径。 对RG4基序的细胞功能的了解与癌症生物学特别相关 和神经退化。含有RG4的RNA区域与含有疾病的区域显著重叠- 相关突变。拟议中的研究可能会阐明正常和 细胞生理学的病理方面，并确定导致肿瘤发生或 神经退行性改变。