Mechanisms Of MicroRNA evolution, regulation and function: computational insight, biological evaluation and practical application MECHANISMS OF MICRORNA EVOLUTION, REGULATION AND FUNCTION: COMPUTATIONAL INSIGHT, BIOLOGICAL EVALUATION AND PRACTICAL APPLICATION Title and Department ____________________________________ Date MECHANISMS OF MICRORNA EVOLUTION, REGULATION AND FUNCTION: COMPUTATIONAL INSIGHT, BIOLOGICAL EVALUATION AND PRACTICAL APPLICATION

Ryan Spengler, Ryan Spengler, Ryan Spengler, Adam, Dupuy, Logsdon John, Andrew, Russo
2013 unpublished
MicroRNAs (miRNAs) are an abundant and diverse class of small, non-protein coding RNAs that guide the post-transcriptional repression of messenger RNA (mRNA) targets in a sequence-specific manner. Hundreds, if not thousands of distinct miRNA sequences have been described, each of which has the potential to regulate a large number of mRNAs. Over the last decade, miRNAs have been ascribed roles in nearly all biological processes in which they have been tested. More recently, interest has grown in
more » ... terest has grown in understanding how individual miRNAs evolved, and how they are regulated. In this work, we demonstrate that Transposable Elements are a source for novel miRNA genes and miRNA target sites. We find that primate-specific miRNA binding sites were gained through the transposition of Alu elements. We also find that remnants of Mammalian Interspersed Repeat transposition, which occurred early in mammalian evolution, provide highly conserved functional miRNA binding sites in the human genome. We also provide data to support that long non-coding RNAs (lncRNAs) can provide a novel miRNA binding substrate which, rather than inhibiting the miRNA target, inhibits the miRNA. As such, lncRNAs are proposed to function as endogenous miRNA "sponges," competing for miRNA binding and reducing miRNAmediated repression of protein-coding mRNA targets. We also explored how dynamic changes to miRNA binding sites can occur by A-to-I editing of the 3 'UTRs of mRNA targets. These works, together with knowledge gained from the regulatory activity of endogenous and exogenously added miRNAs, provided a platform for algorithm development that can be used in the rational design of artificial RNAi triggers with improved target specificity. The cumulative results from our studies identify and in some cases clarify important mechanisms for the emergence of miRNAs and miRNA binding sites on large (over eons) and small (developmental) time scales, and help in translating these gene silencing processes into practical application. ___________________________________ Yi Xing ii ACKNOWLEDGMENTS First of all, I would like to thank my mentor, Dr. Bev Davidson, for her guidance, motivation and most of all, patience. I also must acknowledge all the members of the Davidson Lab, both past and present, who have been an invaluable source of knowledge, discussion and guidance over the years. In particular, I would like to thank Ryan Boudreau and Alex Mas Monteys with whom I have closely collaborated and who are responsible for some of the work presented in this manuscript. I also thank Dr. Anton McCaffrey, my first research mentor, who took the time to train me in the basics of molecular biology techniques. He encouraged me to think outside the box, and guided me as I learned to interpret data and manage my own research projects. I owe a special mention of thanks to the entire faculty in the Biology department of Augustana College, who first taught me to think about science and encouraged me to explore my own interests. Dr. Kristin Douglas and Dr. Dara Wegman-Geedey were particularly amazing mentors who guided me in my own research. Dr. Douglas deserves a special acknowledgement as she first introduced me to microRNAs, which sparked my interest in the subject and led me to follow that interest in my graduate research. Finally, words cannot truly describe my appreciation for the love and support my family has given me over the years. My wife, Erin, most of all has been a vital source of encouragement and the fact that I am writing this manuscript is in large part due to her always being there for me. Thank you all. iii ABSTRACT MicroRNAs (miRNAs) are an abundant and diverse class of small, non-protein coding RNAs that guide the post-transcriptional repression of messenger RNA (mRNA) targets in a sequence-specific manner. Hundreds, if not thousands of distinct miRNA sequences have been described, each of which has the potential to regulate a large number of mRNAs. Over the last decade, miRNAs have been ascribed roles in nearly all biological processes in which they have been tested. More recently, interest has grown in understanding how individual miRNAs evolved, and how they are regulated. In this work, we demonstrate that Transposable Elements are a source for novel miRNA genes and miRNA target sites. We find that primate-specific miRNA binding sites were gained through the transposition of Alu elements. We also find that remnants of Mammalian Interspersed Repeat transposition, which occurred early in mammalian evolution, provide highly conserved functional miRNA binding sites in the human genome. We also provide data to support that long non-coding RNAs (lncRNAs) can provide a novel miRNA binding substrate which, rather than inhibiting the miRNA target, inhibits the miRNA. As such, lncRNAs are proposed to function as endogenous miRNA "sponges," competing for miRNA binding and reducing miRNAmediated repression of protein-coding mRNA targets. We also explored how dynamic changes to miRNA binding sites can occur by A-to-I editing of the 3 'UTRs of mRNA targets. These works, together with knowledge gained from the regulatory activity of endogenous and exogenously added miRNAs, provided a platform for algorithm development that can be used in the rational design of artificial RNAi triggers with improved target specificity. The cumulative results from our studies identify and in some cases clarify important mechanisms for the emergence of miRNAs and miRNA binding sites on large (over eons) and small (developmental) time scales, and help in translating these gene silencing processes into practical application.
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