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

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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.