Dissecting the regulation of RNA sensing in innate immunity

剖析先天免疫中 RNA 传感的调节

• 批准号: 10456287
• 负责人: Wenwen Fang
• 金额: $ 24.9万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10456287
• 关键词: Animals; Biochemical; Biochemistry; Biogenesis; Biological; Cancer Research Project; Cells; Collection; Complex; Coupled; Cryoelectron Microscopy; DNA; Defect; Department of Defense; Development; Disease; Electron Microscopy; Elements; Fellowship; Foundations; Funding; Future; Gene Expression; Generations; Genes; Genome; Goals; Heart; Human; Institutes; Institution; Knowledge; Laboratories; Learning; Malignant Neoplasms; Massachusetts; Mentors; Methods; MicroRNAs; Microprocessor; Modeling; Molecular; Molecular Biology; Molecular Structure; Mutation Analysis; NCOA6 gene; Natural Immunity; Negative Staining; Organism; Oxytricha; Pathway interactions; Phase; Positioning Attribute; Postdoctoral Fellow; Process; Production; Protein Biochemistry; RNA; RNA Binding; RNA Degradation; RNA Interference; RNA-Protein Interaction; Regulation; Regulator Genes; Repression; Research; Research Personnel; Resolution; Resources; Series; Side; Small RNA; Solid; Sterility; Structure; Study Skills; Techniques; Therapeutic; Training; Universities; Ursidae Family; Work; anticancer research; base; carcinogenesis; career; design; experience; genome integrity; insight; interest; malignant breast neoplasm; medical schools; novel; particle; post-doctoral training; pre-doctoral; preservation; protein complex; protein purification; spatiotemporal; structural biology; transcriptome; tumorigenesis

SUMMARY The discovery of RNA interference and related small RNA pathways has revolutionized molecular biology and bears potential for new generations of therapeutics. Endogenous small RNAs such as microRNAs (miRNAs) collectively regulate the majority of the human transcriptome, and control important aspects of spatio-temporal development. Dysregulation of the miRNA pathway therefore directly contributes to developmental defects and tumorigenesis. Another group of small RNAs call Piwi-interacting RNAs (piRNAs) are often active during germline development, and preserve genome integrity by silencing selfish elements such as transposons. Despite of the progress made on understanding small RNA pathways, we still lack a clear picture of how each step of the pathway is carried out. Biochemical and structural characterization of the molecular machineries that promote these steps will represent major advances for the field. I have a long-standing interest in small RNA pathways. During my graduate studies at Princeton University with Dr. Laura Landweber, which were funded by a pre-doctoral fellowship by the Department of Defense Breast Cancer Research Program, I uncovered an unknown function for piRNAs to protect DNA against loss during genome rearrangement in Oxytricha. This was one of the first studies to show “self” recognition by piRNAs, and overturned the dogma that piRNAs only target RNA for repression or DNA for deletion. Then, as a post-doctoral fellow in the laboratory of Dr. David Bartel at the Whitehead Institute, I studied the miRNA pathway, which is the small RNA pathway that dominates in most human cells. Specifically, I identified determinants of primary miRNAs (pri-miRNAs, the precursors of miRNAs) that allow for their efficient processing by the Microprocessor complex, and demonstrated rational, de novo design of artificial miRNA genes. This work was funded by the Damon Runyon Cancer Research fellowship. My long-term goal is to understand how RNA-protein interactions work, with a special focus on small RNA pathways. I would like to pursue this exciting topic as the leader of a research group in an academic institution. To achieve this goal, the overall objectives of this application are to obtain training in protein biochemistry and single-particle cryo-electron microscopy (cryo-EM). This valuable training and experience will lay a solid foundation for me to launch my independent research on RNA-protein complexes. The work proposed here comprises two aims. In Aim 1, which will be completed during the mentored phase, I will leverage my knowledge of pri-miRNAs, the Microprocessor substrate, and harness recent development of cryo-EM, to solve the structure of Microprocessor bound with pri-miRNA, which will provide critical insights into how this complex recognizes its substrate and accurately positions the cleavage. I have already demonstrated that a novel, RNA-based purification strategy allowed the assembly of homogeneous Microprocessor in complex with the substrate, and negative-stain EM shows promising results. My next step is to take advantage of the state-of-the-art cryo-EM technique, which is best suited to solve structures of complexes with a relatively large size. More importantly, this aim will allow me to develop biochemical and structural skills for studying RNA-protein interactions that can be applied to many other RNA-protein complexes, such as the RISC-loading complex proposed in Aim 2. Dr. David Bartel's lab is the ideal place for this training because of its deep expertise in miRNA research, as well as the resources brought in by its affiliation, such as EM facilities at Whitehead Institute, Koch Institute, and the HHMI Research Campus. In Aim 2, which will be the focus of the independent phase, I will fully capitalize the training received in my previous work and that proposed in Aim 1, to solve a long-standing problem at the heart of the small RNA pathways: how does RISC-loading complex work to process pre-miRNA, and help form the mature, silencing-competent RISC complex. Through my previous work, I have obtained extensive molecular biology training. My background and expertise in small RNA pathways ideally positions me for the proposed work. The training and research described in this proposal will allow me to develop a tool-kit of approaches that will form the basis of my independent research, and will provide the platform from which to launch my career as an independent investigator.

总结 RNA干扰和相关小RNA途径的发现彻底改变了分子生物学， 有可能成为新一代的治疗方法内源性小RNA，如microRNA（miRNAs） 共同调节人类转录组的大部分，并控制时空的重要方面。 发展因此，miRNA通路的失调直接导致发育缺陷 和肿瘤发生。另一组称为Piwi相互作用RNA（piRNA）的小RNA通常在细胞周期中活跃。 生殖系发育，并通过沉默自私元件如转座子来保持基因组完整性。 尽管在理解小RNA通路方面取得了进展，但我们仍然缺乏对每个通路如何 该路径的步骤被执行。分子机器的生化和结构表征 推动这些步骤的行动将是该领域的重大进展。 我对小RNA通路有着长期的兴趣。我在普林斯顿读研究生期间 大学与劳拉Landweber博士，这是由博士前奖学金资助的部门 在国防乳腺癌研究计划中，我发现了piRNA保护DNA的未知功能 防止尖毛藻基因组重排过程中的损失。这是最早的研究之一， piRNA的识别，并推翻了piRNA只靶向RNA进行抑制或DNA进行抑制的教条。 删除。然后，作为怀特黑德研究所大卫巴特尔博士实验室的博士后研究员，我 研究了miRNA途径，这是在大多数人类细胞中占主导地位的小RNA途径。 具体来说，我确定了初级miRNAs（pri-miRNAs，miRNAs的前体）的决定因素，这些决定因素允许 他们的有效处理的微处理器复杂，并证明了合理的，从头设计的 人工miRNA基因。这项工作由Damon Runyon癌症研究奖学金资助。 我的长期目标是了解RNA-蛋白质相互作用是如何工作的，特别关注小分子RNA。 RNA途径。我想继续这个令人兴奋的话题，作为一个学术研究小组的领导者， 机构。为了实现这一目标，本申请的总体目标是获得蛋白质方面的培训 生物化学和单粒子冷冻电子显微镜（cryo-EM）。这些宝贵的训练和经验 将为我开展RNA-蛋白质复合物的独立研究奠定坚实的基础。 这里提出的工作包括两个目标。在目标1中，将在受指导期间完成 阶段，我将利用我对pri-miRNAs的知识，微处理器基板，并利用最近的 cryo-EM的发展，以解决与pri-miRNA结合的微处理器的结构，这将提供 关键的见解，这种复合物如何识别其底物和准确定位切割。我有 已经证明，一种新的，基于RNA的纯化策略允许组装同质的 微处理器在复杂的基板，和负染色EM显示有希望的结果。我的下一步是 利用最先进的冷冻EM技术，该技术最适合于解决 具有相对较大尺寸的复合物。更重要的是，这一目标将使我能够开发生物化学和 研究RNA-蛋白质相互作用的结构技巧，可应用于许多其他RNA-蛋白质 复合物，如目标2中提出的RISC负载复合物。大卫·巴特尔博士的实验室是 这项培训是因为它在miRNA研究方面的深厚专业知识，以及它所带来的资源。 这些研究机构包括Whitehead Institute、Koch Institute和HHMI Research Campus的EM设施。在Aim中 2，这将是独立阶段的重点，我将充分利用我以前接受的培训 工作和目标1中提出的，以解决小RNA途径核心的长期问题： RISC装载复合物如何加工pre-miRNA，并帮助形成成熟的，沉默的能力， RISC复合体。 通过我以前的工作，我获得了广泛的分子生物学训练。我的背景和 小RNA途径的专业知识使我非常适合这项工作。培训和研究 本提案中描述的方法将使我能够开发一个方法工具包，这些方法将构成我的基础 独立研究，并将提供平台，从其中启动我的职业生涯作为一个独立的 调查员