Translational Enhancement by tRNA Mimics

tRNA 模拟物的翻译增强

• 批准号: 1411836
• 负责人: Anne Simon
• 金额: $ 74.88万
• 依托单位: University of Maryland, College Park
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1411836&HistoricalAwards=false
• 关键词: Translational; Enhancement; tRNA; Mimics

This project addresses the question of how viruses, which depend on the infected host cells for making virus proteins, use the host cell protein-producing machinery, the ribosome, in a manner that is highly efficient. The research will exploit and extend the novel discovery that the genetic material of a virus contains specific structures that can bind to the ribosomes and that thereby allow them to be quickly and efficiently reused. Key outcomes of this project will be broadly applicable in many fields, including in biotechnology to enhance the efficiency of protein production. The outcomes may also lead to novel approaches to control a variety of viruses that infect plants and animals. This project will provide educational and research training opportunities for high school students, college undergraduates, graduate students and postdoctoral researchers. The outcomes of the research will be presented at scientific conferences, taught in introductory courses to biology students, and used to introduce undergraduates and high school students to the life cycle of viruses. The principal investigator will work with Advanced Placement students at DuVal High School (98% under-represented minority students) to prepare them for college, serve on the board of the National Academy of Sciences partnership with the entertainment industry and will also serve as a scientific consultant for programming by the Discovery and National Geographic Channels.The plant virus "Pea enation mosaic umbravirus" has a bipartite positive-strand RNA genome containing two 3' proximal T-shaped structures (kl-TSS and 3'TSS). These structures are critical for translation of the uncapped viral RNAs into proteins. 3'TSS is an example of the long-known, better-studied tRNA-like structures found in viral RNAs, but the existence of the kl-TSS was unsuspected and came to light during previous research. The newly found, internal kl-TSS, with its three-way branched secondary structure, binds to 40S, 60S and 80S ribosomes and can simultaneously engage in a long-distance interaction with 5H2, a coding region hairpin located near the 5'Untranslated Region (UTR). The 3'TSS is also likely to connect with 5H2, and the whole system will be exploited to learn how different tRNA-mimics interact with ribosomes to enhance translation. Specifically, a novel "pick-up and delivery" hypothesis will be evaluated, i.e., the idea that multiple TSS may be connected via RNA:RNA interactions with the ends of virus-encoded Open Reading Frames (ORF), to gather ribosomal subunits downstream of an ORF at the end of translation and relay them back to the 5'UTR for another round of translation. In addition, it will be tested whether the kl-TSS can enhance translation of capped mRNAs produced from expression constructs. These experiments will inform our understanding of how this novel and possibly widespread use of tRNA mimicry operates to enhance translation and to control plant virus life cycles. The project will also lead to the development of expression cassettes with enhanced translation of encoded proteins with potentially extensive agricultural as well as laboratory applications.

这个项目解决的问题是，依赖于被感染的宿主细胞制造病毒蛋白质的病毒，如何以一种高效的方式利用宿主细胞的蛋白质制造机器——核糖体。这项研究将利用和扩展新的发现，即病毒的遗传物质含有可以与核糖体结合的特定结构，从而使它们能够快速有效地重复使用。该项目的主要成果将广泛应用于许多领域，包括提高蛋白质生产效率的生物技术。这一结果也可能导致控制感染植物和动物的各种病毒的新方法。该项目将为高中学生、大学本科生、研究生和博士后提供教育和研究培训机会。研究结果将在科学会议上发表，在生物学学生的入门课程中教授，并用于向本科生和高中生介绍病毒的生命周期。首席研究员将与杜瓦尔高中（DuVal High School）的大学先修班学生（98%是少数族裔学生）合作，帮助他们为大学做准备，并担任美国国家科学院（National Academy of Sciences）与娱乐行业合作伙伴关系的董事会成员，还将担任探索频道（Discovery）和国家地理频道（National Geographic Channels）节目的科学顾问。植物病毒“豌豆苗花叶伞状病毒”具有双侧正链RNA基因组，包含两个3'近端t形结构（kl-TSS和3ttss）。这些结构对于将无帽病毒rna翻译成蛋白质至关重要。3tss是病毒rna中发现的久已为人所知的trna样结构的一个例子，但kl-TSS的存在是在之前的研究中发现的。新发现的内部kl-TSS具有三向分支二级结构，可结合40S、60S和80S核糖体，并可同时与位于5' untranslanregion （UTR）附近的编码区发夹5H2进行远距离相互作用。3ttss也可能与5H2连接，整个系统将被用来了解不同的trna模拟物如何与核糖体相互作用以增强翻译。具体而言，将评估一种新的“提取和传递”假设，即多个TSS可能通过RNA连接的想法：RNA与病毒编码的开放阅读框架（ORF）末端相互作用，在翻译结束时收集ORF下游的核糖体亚基，并将其传递回5'UTR进行另一轮翻译。此外，还将测试kl-TSS是否能增强表达构建体产生的带帽mrna的翻译。这些实验将使我们了解这种新颖且可能广泛使用的tRNA拟态是如何增强翻译和控制植物病毒生命周期的。该项目还将导致表达盒的发展，增强编码蛋白质的翻译，具有潜在的广泛农业和实验室应用。