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Open Thesis Kayceeaquarles.Pdf The Pennsylvania State University The Graduate School Eberly College of Science THE IMPACT OF PRIMARY MICRORNA STRUCTURE ON RECOGNITION BY THE MICROPROCESSOR COMPLEX IN MICRORNA MATURATION A Dissertation in Chemistry by Kaycee Andrea Quarles © 2015 Kaycee Andrea Quarles Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2015 ii The dissertation of Kaycee Andrea Quarles was reviewed and approved* by the following: Scott A. Showalter Associate Professor of Chemistry Dissertation Advisor Chair of Committee Philip C. Bevilacqua Professor of Chemistry Christine D. Keating Professor of Chemistry Katsuhiko Murakami Associate Professor of Biochemistry and Molecular Biology Barbara J. Garrison Shapiro Professor of Chemistry Head of the Department of Chemistry *Signatures are on file in the Graduate School. iii ABSTRACT Since their discovery over a decade ago, thousands of microRNAs (miRNAs) have been found across all multicellular organisms. These RNAs in combination with small interfering RNAs (siRNAs) make up the RNA silencing pathway, also called the RNA interference (RNAi) pathway. Mature miRNAs are ~22-nucleotide-long, single-stranded non-coding RNAs that participate in various cellular, developmental, and differentiation processes via post- transcriptional regulation of gene expression for more than 90% of human genes. Therefore, these key RNAs have been linked to several disease states including cancer, neurodegenerative diseases, cardiac disease, diabetes, and numerous viral diseases. Recently, they have become a key target for the medical RNA therapeutics community. The human canonical miRNA maturation pathway involves a series of cleavage steps beginning in the nucleus and ending in the cytoplasm, where the final mature miRNA down- regulates gene expression via “silencing” messenger RNA translation. Therefore, these small RNAs down-regulate the expression of every protein within an organism; thus, potentially controlling all bodily processes. The first processing step in the nucleus involves cleavage of the miRNA precursor by the Microprocessor complex, consisting minimally of the RNase III enzyme Drosha and the double-stranded RNA (dsRNA) binding protein DGCR8. The second processing step involves an analogous complex consisting minimally of the RNase III enzyme Dicer and the dsRNA binding protein TRBP. This pathway is unique because all of these mentioned proteins contain dsRNA binding domains (dsRBDs) that help to recognize the miRNA precursors in the cell. However, much is still unknown about the processing of these miRNA precursors into their final mature forms. Although it is known that both Drosha and DGCR8 are required for Microprocessor activity, the molecular mechanism of RNA substrate recognition by these iv proteins is still not fully known. In particular, the means by which Drosha locates and recognizes the exact cleavage site on the RNA plays a critical role in Microprocessor efficiency and must be known to fully understand miRNA biogenesis. Literature suggests that the recognition and cleavage of miRNA precursors is in part based on unique structural characteristics of the RNA, which guide the proteins to their cut-site locations. However, there are currently no experimentally-determined structures of entire miRNA precursors. Therefore, it is necessary to biochemically determine native solution structures of these RNAs as they would be seen by the Microprocessor if the maturation process at the molecular level is to be characterized. Atomic resolution methods for RNA structure determination pose several challenges that are not yet overcome; however, a variety of RNA secondary structure mapping and RNA modeling techniques have proven successful for determining structures of other large RNAs comparable to those encountered in this pathway. Therefore, a primary aim for this thesis was to combine these approaches to analyze structurally diverse miRNA precursors, which revealed a possible Microprocessor recognition site on the RNA. In addition to the recognition site, RNA structure mapping yielded a consistent display of structural deformations periodically placed along the miRNA precursor. Surprisingly, these periodic deformations along miRNA precursors correlate to the binding surface required for dsRBDs, such as those found in both protein components of the Microprocessor complex – while Drosha only contains a single dsRBD, whereas DGCR8 has two in tandem. dsRBDs are characterized as binding to RNA with little sequence specificity; therefore it is reasonable to hypothesize that DGCR8 function is dependent on the recognition of specific structural features in the miRNA precursor. This thesis utilizes a variety of binding techniques to fully characterize the binding of these dsRBDs with RNA, taking into account the different structural features natively present in miRNA precursors. Interestingly, I found that the dsRBD located in Drosha is v not capable of binding RNA at all, leaving DGCR8 as the primary RNA binding protein. Furthermore, DGCR8 showed little sensitivity to the presence of structural deformations within miRNA precursors, leaving us to believe that its tandem dsRBDs are capable of cooperatively binding around them. In the end, while DGCR8 is necessary for dsRNA binding and recruitment to the Microprocessor, it is not sufficient on its own in directing the exact Drosha cut-site position on miRNA precursors. As mentioned, the dsRBDs from Drosha and DGCR8 exhibit very different binding affinities for RNA. The differences seen in RNA binding by dsRBDs from these various proteins begs for an explanation. These dsRBDs display low sequence conservation, which may result in small differences in their folded domains. Therefore, an ongoing aim for this thesis is to examine the structure of dsRBDs bound to dsRNA in order to determine key regions governing the binding interaction as well as portions of the dsRBD important for maintaining the folded domain. However, due to solubility issues, DGCR8 was not amenable to studying this interaction at the atomic level. Instead, results from NMR and X-ray crystallography data of the dsRBD from Dicer (the cytoplasmic processing enzyme) bound to dsRNA will serve as the comparison to bound structures in the Protein Data Bank of dsRBDs with different amino acid sequence. vi TABLE OF CONTENTS LIST OF FIGURES ....................................................................................................................... xii LIST OF TABLES ....................................................................................................................... xvii ACKNOWLEDGEMENTS ........................................................................................................ xviii Chapter 1: Introduction .................................................................................................................... 1 1.1 MicroRNA Maturation Pathway in RNA Interference .......................................................... 1 1.1.1 Primary MicroRNA Structural Recognition by the Microprocessor Complex ............... 4 1.1.2 The Microprocessor Complex of DGCR8 and Drosha ................................................... 8 1.2 RNA Structure Determination ............................................................................................. 12 1.2.1 Atomic Resolution Methods ......................................................................................... 14 1.2.2 Chemical and Enzymatic Probing Methods .................................................................. 16 1.2.3 MC-Pipeline Modeling ................................................................................................. 18 1.3 Double-stranded RNA Binding Domains ............................................................................ 21 1.4 Methods Used for Studying dsRBD Binding ....................................................................... 23 1.4.1 Electrophoretic Mobility Shift Assays .......................................................................... 25 1.4.2 Fluorescence Polarization ............................................................................................. 26 1.4.3 Isothermal Titration Calorimetry .................................................................................. 26 1.4.4 Analytical Ultracentrifugation ...................................................................................... 27 1.4.5 Circular Dichroism ........................................................................................................ 28 1.5 Dissertation Outline ............................................................................................................. 28 1.6 Acknowledgements .............................................................................................................. 29 vii 1.7 References ............................................................................................................................ 30 Chapter 2: The Use of SHAPE Chemistry to Determine RNA Structure in Solution ................... 34 2.1 Introduction .................................................................................................................... 34 2.2 Materials and Methods ................................................................................................... 40 2.2.1 RNA Preparation ........................................................................................................... 40 2.2.2 SHAPE Reagent Preparation .......................................................................................
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