University of Kentucky UKnowledge Theses and Dissertations--Entomology Entomology 2018 THE MECHANISM OF RNA INTERFERENCE IN ARTHROPODS June-Sun Yoon University of Kentucky, [email protected] Digital Object Identifier: https://doi.org/10.13023/ETD.2018.219 Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Yoon, June-Sun, "THE MECHANISM OF RNA INTERFERENCE IN ARTHROPODS" (2018). Theses and Dissertations--Entomology. 45. https://uknowledge.uky.edu/entomology_etds/45 This Doctoral Dissertation is brought to you for free and open access by the Entomology at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Entomology by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. Proper attribution has been given to all outside sources. I understand that I am solely responsible for obtaining any needed copyright permissions. I have obtained needed written permission statement(s) from the owner(s) of each third-party copyrighted matter to be included in my work, allowing electronic distribution (if such use is not permitted by the fair use doctrine) which will be submitted to UKnowledge as Additional File. I hereby grant to The University of Kentucky and its agents the irrevocable, non-exclusive, and royalty-free license to archive and make accessible my work in whole or in part in all forms of media, now or hereafter known. I agree that the document mentioned above may be made available immediately for worldwide access unless an embargo applies. I retain all other ownership rights to the copyright of my work. I also retain the right to use in future works (such as articles or books) all or part of my work. I understand that I am free to register the copyright to my work. REVIEW, APPROVAL AND ACCEPTANCE The document mentioned above has been reviewed and accepted by the student’s advisor, on behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of the program; we verify that this is the final, approved version of the student’s thesis including all changes required by the advisory committee. The undersigned agree to abide by the statements above. June-Sun Yoon, Student Dr. Subba Reddy Palli, Major Professor Dr. Charles W. Fox, Director of Graduate Studies THE MECHANISM OF RNA INTERFERENCE IN ARTHROPODS ____________________________________ DISSERTATION ____________________________________ A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Agriculture, Food and Environment at the University of Kentucky By June-Sun Yoon Lexington, Kentucky Director: Dr. Subba Reddy Palli, Professor of Entomology Lexington, Kentucky Copyright © June-Sun Yoon, 2018 ABSTRACT OF DISSERTATION THE MECHANISM OF RNA INTERFERENCE IN ARTHROPODS RNA interference (RNAi) is a useful reverse genetics tool for investigation of gene function as well as for practical applications in many fields including medicine and agriculture. Due to the variability in RNAi efficiency, RNAi-based methods are currently being developed for controlling only coleopteran insects which are known to be amenable to RNAi. The first chapter of my thesis includes findings from research to investigate what are the factors that make coleopteran insects relatively more efficient in RNAi. I used Colorado potato beetle (CPB), Leptinotarsa decemlineata and its cell line (Lepd- SL1) as study models to identify genes that play key roles in RNAi pathway. Five genes including Argonaute-1 (microRNA Argonaute) and Aubergine (PiwiRNA Argonaute) were identified as those required for siRNA (short interfering RNA) RNAi pathway. I also found that RNAi is completely blocked in StaufenC knockdown cells. StaufenC belongs to dsRNA binding protein family and binds to dsRNA as shown by gel mobility shift and the pull-down assays. Interestingly, I also found that StaufenC is downregulated in RNAi resistant cells and StaufenC homologous sequences are present in only coleopteran insects where RNAi works efficiently. These data suggest that StaufenC is a major contributor to efficient RNAi in coleopteran insects and is a potential target for RNAi resistance. The second part of my research is to understand the mechanisms of RNAi in those insects refractory to RNAi. The barriers for successful RNAi include the presence of double-stranded ribonucleases (dsRNase) in the lumen and hemolymph that could potentially digest double-stranded RNA (dsRNA) and the variability in the transport of dsRNA into and within the cells. Recent work in our laboratory showed that the dsRNAs are transported into lepidopteran cells, but they are not processed into siRNAs because they are trapped in acidic bodies. I focused on identification of these acidic bodies in which dsRNAs accumulate in Spodoptera frugiperda Sf9 cells. These studies showed that entrapment of internalized dsRNA in endosomes is one of the major factors contributing to inefficient RNAi. Overall, my research revealed important players involved in successful and unsuccessful RNAi in insects. KEYWORDS: RNA interference, Mechanism, L. decemlineata, StaufenC, Resistance June-Sun Yoon 06. 04. 2018 THE MECHANISM OF RNA INTERFERENCE IN ARTHROPODS By June-Sun Yoon Subba Reddy Palli Director of Dissertation Charles W. Fox Director of Graduate Studies 06. 04. 2018 Date I would like to dedicate this dissertation to my parents, Sei-Eui Yoon and Sun-Tae Yoo who believed in me and supported me in tremendous ways. Acknowledgments I would like to begin by thanking my advisor, Dr. Subba Reddy Palli, for trusting me and patiently waiting for me to become a decent scientist. I respect him as a brilliant scientist, organized advisor, and sincere mentor. I have enjoyed time discussing with him and will miss those moments. I must also thank my committee Dr. Daniel Potter who have supported me mentally and ‘Fish’ically from the day one of my graduate life. Dr. Xuguo Zhou always provides the wise suggestions to improve my scientific challenges. Dr. Michael Goodin and his lab member ChanYong Jang helped me to understand and performed plant-based experiments. I appreciated Dr. John Obrycki and Dr. Chuck Fox for taking care of students and providing me professional or personal advice with a bright smile. Moreover, I would like to thank all the faculty members in this department because they have each guided me at one point or another. I would like to express sincere appreciation to my current and previous lab members. I have asked more than 1000 questions to Dr. Jayendra Shukla, and he has sincerely answered every single question based on his wisdom and knowledge. You are awesome, Dr. Jayendra Shukla. Dr. Jingjing Xu was always willing to help and teach me how to design the decent experiments. Dr. Megha Kalsi was a “frenemy” who was both a good lab member and the enemy who pushed me to better at the same time. Smitha George is a smart colleague who always helps to organize the lab. Dhandapani Gurusamy, Kanakachari Mogilicherla, Dr. Ramesh Kumar Dhandapani are my collaborators for intellectual discussions and constructive criticism. Jeff Howell is the best technician ever who shared all my burden from the lab duties. I also want to thank Brian Lauer for all the technical support. I would also like to take time to thank all my iii people in this department who shared the harsh time and happy moments. I would especially like to thank Emrah Ozel, Josiah Ritchey, Amanda Skidmore, Thor Hanson, Caitlin Stamper, Paul Baker, Lauren Fann, Jinmo Koo, Yaoyu Jiao, and Izabela Gomes. Finally, I would like to express my gratitude to my friends outside the department. Kyungbo Kim is another brilliant scientist who solves my scientific thirsty and encourages me when I was depressed with depressing results. Thank all my besties from WKU/Nashville crews. I would not have accomplished anything today without my parents’ support and trust me. iv TABLE OF CONTENTS ACKNOWLEDGMENTS ................................................................................................ III TABLE OF CONTENTS ................................................................................................... V LIST OF TABLES ......................................................................................................... VIII LIST OF FIGURES .......................................................................................................... IX CHAPTER 1. LITERATURE REVIEW ........................................................................ 1 RNA interference ............................................................................................................................................ 1 RNAi pathways ........................................................................................................................................... 1 Mechanism of RNAi ................................................................................................................................... 2 RNAi in insects ............................................................................................................................................... 2 Factors responsible for variable RNAi efficiency among insects .................................................................... 3 Delivery methods .......................................................................................................................................