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：
10058846
负责人：
Zhipeng Lu
金额：
$ 29.5万
依托单位：
UNIVERSITY OF SOUTHERN CALIFORNIA
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-12-17 至 2021-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10058846
关键词：
Address Atlases Base Pairing Binding Proteins Binding Sites Biochemical Carbazoles Catalysis Cells Chemicals Clinical Collection Communities Complex Crosslinker Crystallography DNA-Protein Interaction Data Detection Disease Elements Engineering Enzymes Exonuclease Foundations Gel Gene Expression Process Gene Expression Regulation Genetic Genetic Translation Genome Genomics Goals Health High-Throughput Nucleotide Sequencing Human Infection Instruction K-Series Research Career Programs Lead Ligation Malignant Neoplasms Medicine Messenger RNA Methods Molecular Conformation Mutagenesis Mutation Nucleotides Oligonucleotides Paris, France Phenotype Phylogenetic Analysis Physiological Proteins Psoralens RNA RNA Helicase RNA Virus Infections RNA Viruses RNA analysis RNA-Binding Proteins Reporter Research Resolution Role Sampling Solubility Speed Structural Models Structure Testing Translating Translations Untranslated RNA Validation animal tissue base computerized tools crosslink design experimental study flexibility genetic information genomic RNA helicase human disease in vivo insight method development recruit scaffold screening single molecule success tool transcriptome viral genomics

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. PARIS-determined structures 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. In summary, the proposed studies will deliver a set of powerful tools to the broad RNA community for RNA structure determination, 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.

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 结构代表了重要但未被充分认识的遗传信息层。对于基因组蓝图的解释和执行至关重要。在这份职业发展奖申请中，我概述了从开发 RNA 结构测定方法到 RNA 结构功能表征的研究策略，这些策略最终将应用于人类。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，RNA 结构或解旋酶（重塑 RNA 结构的酶）的遗传改变是许多人类疾病的基础，包括各种癌症。最致命的人类感染，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，我开发了 PARIS。基于补骨脂素交联的方法，可在单分子水平上以接近碱基对的分辨率对活细胞中的 RNA 双链体进行高通量作图（Lu et al. 2016 Cell）。在本提案中，我将通过开发新型高效光化学交联剂（高溶解度补骨脂素和双功能咔唑）和 PARIS 确定的分辨率优化酶结构来进一步增强 PARIS 的能力，从而可以使用 PARIS 对其功能进行全局筛选。合成翻译生产。系统诱变和蛋白质效应子筛选将用于剖析调节结构元件的功能机制。总之，拟议的研究将为广泛的 RNA 界提供一套用于 RNA 结构测定的强大工具，并通过对 RNA 结构的多尺度询问（从结构域到单碱基对的全面表征）提供对 RNA 功能的新见解。 RNA结构组将揭示在生理和疾病背景下将基因组转化为表型的调控机制。

项目成果

期刊论文数量（4）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

RISE: a database of RNA interactome from sequencing experiments.

RISE：来自测序实验的 RNA 相互作用组数据库。

DOI：
发表时间：
2018
期刊：
影响因子：
14.9
作者：
Gong, Jing;Shao, Di;Xu, Kui;Lu, Zhipeng;Lu, Zhi John;Yang, Yucheng T;Zhang, Qiangfeng Cliff
通讯作者：
Zhang, Qiangfeng Cliff

Mechanistic insights in X-chromosome inactivation.

X 染色体失活的机制见解。