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

该奖项是根据2009年美国复苏和再投资法案（公法111-5）资助的。在美国，由于植物病原体（包括植物病毒）感染而造成的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增加受感染植物中症状严重性的小病毒相关（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，这可以进一步增强这些病原体的感染性。 RNA病毒学领域的一个主要问题仍然是“是什么使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、在宿主中积累并引起疾病的能力有关。 芜菁皱缩病毒（Turnip crinkle virus，TCV）的亚病毒RNA卫星C（satC）由于其小的尺寸，是鉴定和表征参与复制和致病的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）是一种方法，其中一部分satC RNA被随机化，然后在TCV存在下使用这些随机化的satC RNA的池来感染植物。 只有功能性的satC分子在植物中移动，并且可以在新叶中回收;因此，可以鉴定这些satC中所需的RNA序列和/或结构。 通过这种在satC的5 ′部分内去进化和再进化不同元件的方法，将定义控制（i）从（-）-链复制中间体合成satC（+）-链和（ii）satC单体与二聚体之比的信号。 在satC 5 ′端定义序列/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名本科生将通过在迪金森学院的RNA生物学课程的两个学期的实验室练习中启动研究项目来为研究做出贡献;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BIO 2010指南中所建议的）。 此外，这项研究将根据生物学和生物化学分子生物学学生专业的当前人口统计数据（可能参加该课程）以及新的（迪金森科学波塞; NSF DUE-0856704）努力，以加强对自然科学感兴趣的少数民族的招聘和保留。 在主要研究者的研究实验室从事该项目的本科生将出席全国会议，以便他们能够在提高研究意识的同时为研究文化做出贡献。 学生还将有助于研究出版物的写作。 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。

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 lab 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.