Collaborative Research/RUI: Identification of cis-acting sequence and structural elements required for replication of a viral RNA

合作研究/RUI：鉴定病毒 RNA 复制所需的顺式作用序列和结构元件

• 批准号: 0918624
• 负责人: David Kushner
• 金额: $ 15.72万
• 依托单位: Dickinson College
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-12-01 至 2014-11-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0918624&HistoricalAwards=false
• 关键词: Collaborative; Research; RUI; Identification; cis

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).In the United States, crop loss due to infection by plant pathogens, including plant viruses, has been estimated to exceed thirty billion dollars per year. In the environment, the genomes of plant viruses, as well as small, virus-associated (subviral) RNAs that often augment the severity of symptoms in infected plants, can exchange genetic information by recombination and/or rapidly self-evolve, which can further enhance infectiousness of these pathogens. A major question in the field of RNA virology remains: "What makes an RNA infectious?" One thought is that the viral RNA sequence and/or structure that the RNA forms relates to its ability to replicate its genetic material, accumulate in its host, and cause disease. Due to its small size, the subviral RNA satellite C (satC) of Turnip crinkle virus (TCV) is an excellent model for identification and characterization of RNA sequences and/or structures involved in replication and pathogenesis. In vivo SELEX (systematic evolution of ligands by exponential enrichment) is a method in which a portion of the satC RNA is randomized, and then pools of these randomized satC RNAs are used to infect plants in the presence of TCV. Only functional satC molecules move through the plants and can be recovered in new leaves; therefore, required RNA sequences and/or structures in these satC can be identified. Through this approach of de-evolving and re-evolving different elements within the 5´ portion of satC, signals that control (i) satC (+)-strand synthesis from (-)-strand replication intermediates and (ii) the ratio of satC monomers to dimers will be defined. Defining sequence/structure requirements in the satC 5´ end will enhance our current understanding of how satC functions in TCV infection.Broader impacts. About 30 undergraduates will contribute to the research - most via initiating the research projects during semester-long laboratory exercises in two offerings of an RNA Biology course at Dickinson College; others will complete the studies in the principal investigator's research lab. The students will answer state-of-the-art questions in RNA biology and virology by learning molecular biology techniques and performing in-depth data analysis, therefore partaking in a true research experience (as recommended in the BIO2010 guidelines). Furthermore, this research will engage women and underrepresented minorities based on current demographics of student majors in Biology and Biochemistry & Molecular Biology (likely participants in the course) as well as new (Dickinson science posse; NSF DUE-0856704) efforts to enhance recruitment and retention of minorities with interests in the Natural Sciences. The undergraduates who work on this project in the principal investigator's research laboratory will present at national meetings so that they can contribute to the research culture while enhancing their research awareness. The students also will contribute to the writing of research publications. Additionally, the principal investigators will write an education paper in order to disseminate the concept of in vivo SELEX experiments as a pedagogical tool for open-ended, molecular biology research projects for undergraduates.

该奖项根据 2009 年美国复苏和再投资法案（公法 111-5）提供资金。在美国，由于植物病原体（包括植物病毒）感染造成的农作物损失估计每年超过 300 亿美元。 在环境中，植物病毒的基因组以及通常会加重受感染植物症状严重程度的小型病毒相关（亚病毒）RNA，可以通过重组和/或快速自我进化来交换遗传信息，从而进一步增强这些病原体的传染性。 RNA病毒学领域的一个主要问题仍然存在：“是什么让RNA具有传染性？” 一种想法是，病毒 RNA 序列和/或 RNA 形成的结构与其复制遗传物质、在宿主体内积累和引起疾病的能力有关。 由于其体积小，芜菁皱病毒 (TCV) 的亚病毒 RNA 卫星 C (satC) 是识别和表征参与复制和发病机制的 RNA 序列和/或结构的极好模型。 体内SELEX（通过指数富集的配体系统进化）是一种将一部分 satC RNA 随机化的方法，然后使用这些随机化 satC RNA 池在 TCV 存在的情况下感染植物。 只有功能性 satC 分子才能穿过植物并在新叶中恢复；因此，可以鉴定这些 satC 中所需的 RNA 序列和/或结构。 通过这种对 satC 5´ 部分内的不同元件进行去进化和再进化的方法，将定义控制 (i) satC (+)-链从 (-)-链复制中间体合成和 (ii) satC 单体与二聚体比率的信号。 定义 satC 5' 端的序列/结构要求将增强我们目前对 satC 在 TCV 感染中如何发挥作用的理解。更广泛的影响。 大约 30 名本科生将为这项研究做出贡献，其中大部分是通过在迪金森学院的两门 RNA 生物学课程中为期一个学期的实验室练习中启动研究项目；其他人将在首席研究员的研究实验室完成研究。 学生将通过学习分子生物学技术和进行深入的数据分析来回答 RNA 生物学和病毒学中最先进的问题，从而参与真正的研究体验（如 BIO2010 指南中所建议的那样）。 此外，这项研究将根据生物学和生物化学与分子生物学专业（可能参加课程）的学生当前人口统计数据，以及新的（狄金森科学团队；NSF DUE-0856704）努力，吸引女性和代表性不足的少数群体，以加强对自然科学感兴趣的少数群体的招募和保留。 在首席研究员研究实验室从事该项目的本科生将在全国会议上发言，以便他们能够为研究文化做出贡献，同时提高他们的研究意识。 学生还将为研究出版物的写作做出贡献。 此外，主要研究人员将撰写一篇教育论文，以传播体内 SELEX 实验的概念，作为本科生开放式分子生物学研究项目的教学工具。