Decoding the RNA structurome: method development and function analysis

解码 RNA 结构组：方法开发和功能分析

基本信息

批准号：
9369932
负责人：
Zhipeng Lu
金额：
$ 13.17万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-08-15 至 2019-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9369932
关键词：
Address Atlases Base Pairing Binding Proteins Binding Sites Biochemical Biochemistry Biology Carbazoles Catalysis Cells Chemicals Chromatin Clinical Collaborations Collection Communities Complex Computer Analysis Crosslinker Crystallography DNA-Protein Interaction Data Detection Disease Elements Engineering Environment Enzymes Exonuclease Faculty Ficusin Foundations France Future Gel Gene Expression Process Gene Expression Regulation Genetic Genetic Translation Genome Genomics Goals Growth and Development function Health Hearing High-Throughput Nucleotide Sequencing Human Infection Institutes Instruction International Israel K-Series Research Career Programs Lead Ligation Malignant Neoplasms Medicine Mentors Messenger RNA Methods Modeling Molecular Conformation Mutagenesis Mutation Nucleotides Oligonucleotides Phenotype Photochemistry Phylogenetic Analysis Physiological Positioning Attribute Proteins Psoralens RNA RNA Helicase RNA Virus Infections RNA Viruses RNA analysis RNA-Binding Proteins Recruitment Activity Reporter Research Research Institute Resolution Role Sampling Site Solid Solubility Speed Structure Testing Training Translating Translations Untranslated RNA Validation Viral Work X Inactivation animal tissue base career career development computerized tools crosslink design experimental study flexibility genetic information genomic RNA helicase high throughput screening human disease in vivo insight member method development programs scaffold screening single molecule skills stem cell biology success support network tool transcriptome

项目摘要

Project Summary The long-term goal of my research is to comprehensively characterize the RNA structurome, here defined as the collection of all RNA structures and RNA-RNA interactions in living cells. RNA structures represent an important, yet under-appreciated, layer of genetic information that is essential for the interpretation and execution of the genomic blueprint. In this application for career development award, I have outlined research strategies that range from development of methods for RNA structure determination to functional characterization of RNA structures, which will eventually lead to applications to human diseases, the ultimate goal of genome medicine. RNA structures and interactions are fundamental to RNA's diverse functions, such as guiding, scaffolding and catalysis. Not surprisingly, genetic alternations of RNA structures or helicases (enzymes that remodel RNA structures) underlie many human diseases, including various cancers. RNA viruses cause some of the most deadly human infections, and viral genomic RNA structures control critical steps in their lifecycle. However, only a few RNA structures have been determined due to lack of proper methods. To address this issue, I developed PARIS, a psoralen-crosslinking based method for high throughput mapping of RNA duplexes in living cells at single-molecule level with near base-pair resolution (Lu et al. 2016 Cell). In this proposal, I will further increase the capabilities of PARIS by developing new high-efficiency photochemical crosslinkers (high solubility psoralens and bifunctional carbazoles) and resolution-refining enzymatic strategies. Based on the PARIS-determined structures, and integrating clustered interaction sites of RNA-binding proteins, I will test the hypothesis that RNA domains are structural and functional units of long non-coding RNAs. PARIS-determined structures also made it possible to conduct global screens for their functions using synthetic translation reporters. Systematic mutagenesis and screening of protein effectors will be used to dissect the mechanism of function for regulatory structural elements. My previous training in a wide variety of topics that span computational and experimental biology set a solid foundation for my future success. To achieve my career goals goals, I have designed further training plans that will equip me with the critical skills in chemical biology, computational analysis and high throughput function screening. On one hand, I will obtain training through collaborations with Stanford faculty members, including my mentor Dr. Howard Chang an expert in chromatin and RNA biology, Dr. Eric Kool, an expert in photochemistry, Dr. Rhiju Das, an expert in computational biochemistry and RNA structure analysis, Dr. Peter Sarnow, an expert in translational control and RNA virus. The work conducted at Stanford will be well supported by the superb academic environment that includes all the intellectual interactions and necessary facilities. On the other hand, my team of collaborators includes international experts such as Dr. Edith Heard from the Curie Institute in France, who specializes in stem cell biology and X chromosome inactivation, and Dr. Eran Segal at the Weizmann Institute in Israel, who specializes in genomics and high throughput screens. Together, my support network provides me with the essential assistance for both scientific growth and career development. In summary, the proposed studies will deliver a set of powerful tools to the broad RNA community for RNA structure determination, elaborate and validate the new concept of high-level RNA modularity, and provide new insights into RNA functions through multi-scale interrogation of RNA structures, from domains to single base pairs. Comprehensive characterization of the RNA structurome will uncover the regulatory mechanisms that translate the genome to phenotype, in both physiological and disease contexts. This training plan will not only establish a successful research program but will also facilitate my transition to an independent faculty position in a top research institute.

项目概要我研究的长期目标是全面表征 RNA 结构组，这里定义为活细胞中所有RNA结构和RNA-RNA相互作用的集合。 RNA结构代表了一个重要但未被充分认识的遗传信息层，它对于基因组蓝图的解释和执行。在这份职业发展奖申请中，我有概述了研究策略，范围从开发 RNA 结构测定方法到 RNA结构的功能表征，最终将应用于人类疾病基因组医学的最终目标。 RNA 结构和相互作用是 RNA 多种功能的基础，例如引导、支架和催化。毫不奇怪，RNA 结构或解旋酶（解旋酶）的遗传改变重塑RNA结构）是许多人类疾病的根源，包括各种癌症。 RNA病毒会引起一些病毒基因组 RNA 结构控制着其生命周期的关键步骤。然而，由于缺乏适当的方法，仅确定了少数RNA结构。为了解决这个问题问题，我开发了 PARIS，一种基于补骨脂素交联的方法，用于高通量 RNA 作图活细胞中单分子水平的双链体，具有接近碱基对的分辨率（Lu et al. 2016 Cell）。在本提案中，我将通过开发新的高效能进一步增强 PARIS 的能力光化学交联剂（高溶解度补骨脂素和双功能咔唑）和分辨率提高酶促策略。基于PARIS确定的结构，并整合聚集的相互作用位点 RNA 结合蛋白，我将检验以下假设：RNA 结构域是长链的结构和功能单位非编码RNA。巴黎确定的结构也使得对其进行全球筛选成为可能使用合成翻译记者的功能。蛋白质效应子的系统诱变和筛选将用于剖析调节结构元件的功能机制。我之前接受的涵盖计算和实验生物学的各种主题的培训为我奠定了基础为我未来的成功奠定了坚实的基础。为了实现我的职业目标，我设计了进一步的培训计划将使我具备化学生物学、计算分析和高通量方面的关键技能功能筛选。一方面，我将通过与斯坦福大学教员的合作获得培训，包括我的导师 Howard Chang 博士（染色质和 RNA 生物学专家）、Eric Kool 博士（染色质和 RNA 生物学专家）光化学，Rhiju Das 博士，计算生物化学和 RNA 结构分析专家，Peter 博士 Sarnow，翻译控制和RNA病毒方面的专家。在斯坦福大学进行的工作将会很好由一流的学术环境支持，包括所有智力互动和必要的设施。另一方面，我的合作者团队包括国际专家，例如 Edith Heard 博士来自法国居里研究所，专门研究干细胞生物学和 X 染色体失活，博士。以色列魏茨曼研究所的 Eran Segal，专门研究基因组学和高通量筛选。我的支持网络共同为我的科学成长和职业生涯提供了必要的帮助发展。总之，拟议的研究将为广泛的 RNA 界提供一套强大的工具，用于 RNA结构测定，阐述并验证高级RNA模块化的新概念，以及通过对 RNA 结构（从结构域到结构域）的多尺度询问，为 RNA 功能提供新的见解单碱基对。 RNA 结构组的全面表征将揭示调控机制在生理和疾病背景下将基因组转化为表型的机制。本次培训计划不仅将建立一个成功的研究计划，而且还将促进我向一个顶尖研究机构的独立教职。