Dissecting the regulation of RNA sensing in innate immunity

摘要 RNA干扰和相关小RNA途径的发现使分子生物学和 蕴含着新一代疗法的潜力。内源性小RNA，如microRNAs(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途径的失调直接导致发育缺陷 和肿瘤的发生。另一组称为Piwi相互作用RNA(PiRNAs)的小RNA通常在 生殖系发育，并通过沉默转座子等自私成分来保持基因组的完整性。 尽管在理解小RNA途径方面取得了进展，但我们仍然缺乏一个清楚的图景来了解每条途径是如何 执行路径的步骤。分子机械的生物化学和结构表征 促进这些步骤将是该领域的重大进展。 长期以来，我一直对小RNA途径感兴趣。我在普林斯顿大学读研究生期间 与劳拉·兰德韦伯博士合作的大学，由博士后奖学金资助 防御性乳腺癌研究计划，我发现了piRNAs保护DNA的未知功能 防止棘毛虫基因组重排过程中的丢失。这是第一批显示“自我”的研究之一。 被piRNAs识别，并推翻了piRNAs只针对RNA进行抑制或针对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。 具体地说，我确定了主要miRNAs的决定因素(pri-miRNAs，miRNAs的前体)，允许 它们的高效处理由微处理器复合体完成，并展示了合理的从头设计 人造miRNA基因。这项工作是由达蒙·鲁尼恩癌症研究奖学金资助的。 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途径。作为一个学术研究小组的组长，我想继续这个令人兴奋的话题 机构。为了实现这一目标，该应用程序的总体目标是获得蛋白质方面的培训 生物化学和单颗粒低温电子显微镜(Cryo-EM)。这次宝贵的培训和经验 将为我开展RNA-蛋白质复合体的自主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，将在指导期间完成 在这个阶段，我将利用我对pri-miRNAs、微处理器基板的知识，并利用最近 开发了Cryo-EM，解决了与pri-miRNA结合的微处理器的结构，将提供 对这种复合体如何识别其底物并准确定位乳沟的关键见解。我有过 已经证明了一种新的、基于RNA的纯化策略允许组装同源 微处理器在与底物的络合和负染EM方面显示出可喜的结果。我的下一步是 利用最先进的低温电磁技术，它最适合于解决 具有较大规模的复合体。更重要的是，这个目标将使我能够发展生化和 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 复合体，如目标2中建议的RISC负载复合体。David Bartel博士的实验室是 这一培训是因为它在miRNA研究方面的深厚专业知识，以及其带来的资源 隶属关系，例如怀特黑德研究所、科赫研究所和HHMI研究园区的EM设施。在AIM 2，这将是独立阶段的重点，我将充分利用我在上一个阶段接受的培训 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负载复合体如何处理前miRNA，并帮助形成成熟的、沉默能力强的 RISC复合体。 通过我以前的工作，我获得了广泛的分子生物学培训。我的背景和 在小RNA途径方面的专业知识使我能够胜任拟议中的工作。培训和研究 这份建议书中所描述的将使我能够开发一个方法工具包，这些方法将构成我的 独立研究，并将提供一个平台，从那里开始我的独立职业生涯 调查员。

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.