Decoding the RNA Structurome: Method Development and Function Analysis.

项目摘要 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的，但未得到充分重视的遗传信息层，这对于解释和执行基因组蓝图至关重要。在这个职业发展奖的申请中，我概述了从RNA结构测定方法的开发到RNA结构的功能表征的研究策略，这些策略最终将导致应用于人类疾病，这是基因组医学的最终目标。 RNA的结构和相互作用是RNA的多种功能的基础，如引导，支架和催化。毫不奇怪，RNA结构或解旋酶（重塑RNA结构的酶）的遗传变异是许多人类疾病（包括各种癌症）的基础。RNA病毒引起一些最致命的人类感染，病毒基因组RNA结构控制着它们生命周期中的关键步骤。然而，由于缺乏合适的方法，只有少数RNA结构被确定。为了解决这个问题，我开发了PARIS，这是一种基于peptide交联的方法，用于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，我将通过开发新的高效光化学交联剂（高溶解度peptides和双功能咔唑）和分辨率精炼酶策略来进一步提高PARIS的能力。PARIS确定的结构使得使用合成翻译报告子对其功能进行全局筛选成为可能。系统诱变和蛋白质效应物的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社区提供一套强大的工具，用于RNA结构测定，并通过对RNA结构的多尺度询问（从结构域到单碱基对）提供对RNA功能的新见解。RNA结构组的全面表征将揭示在生理和疾病背景下将基因组翻译为表型的调控机制。

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.