Fluorescent Labeling, Co-Tracking, and Quantification of RNA In Cellulo by Thomas Corey Custer A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Chemical Biology) in the University of Michigan 2016 Doctoral Committee: Professor Nils G. Walter, Chair Professor Daniel Goldman Associate Professor Bruce Palfey Assistant Professor Sarah Veatch ©Thomas Corey Custer 2016 DEDICATION I hereby dedicate this body of work to my wife and son, Danielle & Abel Custer, and my parents, Thomas & Becky Custer. ii ACKNOWLEDGMENTS I would like to thank all of my friends, family and colleagues for your support over the years. Every contribution you have made to my life, no matter the magnitude, helped shape who I am and what I have accomplished. To my Mom and Dad (Thomas & Becky Custer), thank you for your love and encouragement, not to mention all of your financial and emotional contributions. There were many times in my life I felt alone, defeated, without direction and helpless; you both provided me the resources, the strength and encouragement that pushed me to become someone of substance, in the eyes of God. Sincerely, thank you. To my wife Danielle Custer, none of this would be possible without you. You were my strength when I was weak, my confidant when I needed to unburden myself, my inspiration when I was disheartened, my joy when I was sad, and my friend when I felt alone. Thank you for everything, but most of all, thank you for being the mother to our beautiful son, Abel. You are as deserving of recognition for this body of work as I am. Together, we will always succeed. I love you so much and am blessed to have you in my life. iii TABLE OF CONTENTS DEDICATION ii ACKNOWLEDGMENTS iii LIST OF FIGURES vii LIST OF TABLES x ABSTRACT xi CHAPTER I. Under the Microscope: A Historical Overview of Single-Molecule Approaches and Analyses of Intracellular RNA 1 1.1 Introduction 1 1.2 Cell Biology of RNA 2 1.3 Principles of Intracellular Single Molecule Fluorescence Microscopy of RNA 20 1.4 Recent Applications of Single Molecule Approaches to RNA in Cellulo 76 1.5 Summary 92 1.6 Thesis Overview 93 II. Testing the miR-21 Target Engagement Hypothesis: Comparing Immortal and Primary 97 2.1 Introduction 97 2.2 Materials and Methods 101 iv 2.3 Results 107 2.4 Discussion 129 2.5 Acknowledgements 132 III. Designing and Labeling Long RNA for Intracellular Single-molecule Fluorescence Microscopy 133 3.1 Introduction 133 3.2 Materials and Methods 136 3.3 Results 153 3.4 Discussion 178 3.5 Acknowledgements 186 IV. Intracellular Behaviors of Fluorescent mRNA and Pseudogenes 187 4.1 Introduction 187 4.2 Materials and Methods 193 4.3 Results 200 4.4 Discussion 218 4.5 Acknowledgements 220 V. Gene-Actin Tethered Intracellular Co-tracking Assay (GATICA) 5.1 Introduction 221 5.2 Materials and Methods 221 5.3 Results 225 5.4 Discussion 231 VI. Summary and Future Outlook 236 6.1 Summary 236 v 6.2 Future Outlook 240 REFERENCES 245 vi List of Figures Figure 1.1: Survey of the RNA biology in a eukaryotic cell 6 Figure 1.2: Photophysical properties of fluorophores 30 Figure 1.3: Fluorescently labeling RNA by hybridization of labeled probes 37 Figure 1.4: Recent techniques for fluorescently labeling RNA by hybridization of labeled probes 44 Figure 1.5: RNA labeling by various protein-RNA tethering approaches 49 Figure 1.6: Chemical and enzymatic methods for direct fluorophore labeling RNA 57 Figure 1.7: Schematic of our home-built single molecule microscope 63 Figure 1.8. Various types of illumination geometries 67 Figure 1.9: Transcriptional bursting measured by smFISH in mammalian cells 80 Figure 1.10. Cytoplasmic mRNP dynamics 86 Figure 1.11: iSHiRLoC of miRNAs 90 Figure 2.1: Intracellular single particle tracking of Alexa-647 labeled miR-21 in HeLa cells 113 Figure 2.2: Intracellular single particle tracking of Alexa-647 labeled miR-21 in PMC KO 114 Figure 2.3: Let-7a-1 live-cell particle tracking in HeLa cells 116 Figure 2.4. Co-tracked Alexa 647 labeled miR-21 with p-bodies in DCP1a-EGFP stably transfected U2OS cells 118 vii Figure 2.5. iSHiRLoC processes do not induce, nor do miR-21 largely enter, stress Granules 122 Figure 2.6. Chemical inhibitors of translation do not express the appropriate phenotype in HeLa cells 124 Figure 2.7. Fluorescent miRNA Colocalize with Lysosome Marker in RISC Independent Fashion 125 Figure 2.8. Endogenous and exogenous miR-21 function to repress genes in a miRNA and siRNA capacity in both mouse primary and cancer cell lines 127 Figure 3.1: Design of dual luciferase reporter and T7 promoter containing pcDNA3 (-) plasmid systems 154 Figure 3.2: Luciferase repression assays of dual luciferase plasmid constructs containing 0 – 6 & 11 miR-7 MRE, with various affinities to miR-7, on the FLuc gene 158 Figure 3.3: Luciferase repression assays of dual luciferase plasmid constructs containing 0 – 6 & 11 miR-21 MRE, with various affinities to miR-21, on the FLuc gene 160 Figure 3.4: Artificial FLuc pseudogene do not code for protein 164 Figure 3.5: Labeling schemes of the mRNA 167 Figure 3.6: Extent of labeling and post-transcriptional RNA modification for all three labeling strategies 168 Figure 3.7: Copper based click chemistry approach degrades BBT modified RNA 171 Figure 3.8: BBT & Tail Modified mRNA produce translate protein 174 viii Figure 3.9: Microinjected labeled BBT and Tail Modified mRNA selectively express FLuc protein 175 Figure 3.10: Fluorescent BBT and Tail Modified FLuc RNA are repressed and degraded by miRNA 179 Figure 3.11: Cy5-body labeled mRNA colocalize with P-Body marker DCP1a- EGFP 182 Figure 4.1: Quantitative modelling of competition effects for miR-20a binding 190 Figure 4.2: Calibrating amounts of RNA injected 202 Figure 4.3: Single – molecule analysis of fluorescent mRNA in cellulo 206 Figure 4.4: 3’ fluorescently labeled miRNA will repress a luciferase reporter in cellulo 210 Figure 4.5: Fixed cell analysis of Microinjected Cy5-body labeled FLuc RNA, containing 11 miR-7 MRE, and 3’ labeled Cy3-miR-7 guide strand duplexed with 5’ Iowa Black® RQ labeled passenger strand 212 Figure 4.6: Cy5-UTP and partially digested RNA half-lives are close to 4 h 214 Figure 4.7: Degraded RNA diffusion coefficients are indistinguishable from FLuc pseudogene and coding genes 216 Figure 5.1: Gene-Actin Tethered Intracellular Co-tracking Assay (GATICA) 227 Figure 5.2: Fluorescent RNA are tethered in a streptavidin dependent manner 228 Figure 5.3: Fluorescent miRNA seldom are found associated with tethered RNA target 232 Figure 5.4: Biotinylated-actin diffuse more rapidly than phalloidin tethered target RNA 233 ix List of Tables Table 1.1. Representative classes of RNAs found in a eukaryotic cell 22 Table 1.2. Nuclear diffusion characteristics of RNAs of varying length 82 Table 2.1: The diffusion coefficient area under the curve for each miR-21 subpopulation, for select time-points 115 Table 2.2. The extent of colocalization of microinjected Alexa-647 labeled miR-21 with DCP1a-EGFP cytoplasmic foci in U2OS cells. 119 Table 3.1: Primer list for cloning pmiR-Glo and pcDNA3.1 (-) miR-7 3’UTR containing constructs 145 Table 3.2: Primer list for cloning pmiR-Glo and pcDNA3.1 (-) miR-21 3’UTR containing constructs 149 Table 3.3: Calculated parameters for each labeling strategy 172 Table 4.1: Calculated Diffusion parameters for each labeling strategy 213 x Abstract Fluorescent Labeling, Co-Tracking, and Quantification of RNA In Cellulo by Thomas Corey Custer Chair: Nils G. Walter RNA plays a fundamental, pervasive role in cellular physiology, through the maintenance and controlled readout of all genetic information, a functional landscape we are only beginning to understand. In particular, the cellular mechanisms for the spatiotemporal control of the plethora of RNAs are still poorly understood. Intracellular single-molecule fluorescence microscopy provides a powerful emerging tool for probing the pertinent biophysical and biochemical parameters that govern cellular RNA functions, including those of protein-encoding mRNAs. Yet progress has been hampered by the scarcity of high-yield, efficient methods to fluorescently label RNA molecules without the need to drastically increase their molecular weight through artificial appendages that may result in altered behavior. Herein, we employ a series of in vitro enzymatic techniques to efficiently, extensively and in high-yield, incorporate chemically modified nucleoside triphosphates into a transcribed messenger RNA body, between its body and tail (BBT), or randomly throughout the poly(A) tail (tail). Of these, BBT and tail modified strategies proved the most promising methods to functionally label messenger RNA and single- particle track their behaviors using our in-house single-molecule assay: intracellular xi single-molecule high resolution localization and counting (iSHiRLoC). From this research also was spawned a novel method to anchor an RNA to the actin cytoskeleton for the study of long-term interactions within a cellular context, termed: Gene-Actin Tethered Intracellular Co-tracking Assay (GATICA). Here, biotinylated RNA is tethered to the actin surface, either through complexation with a streptavidin coupled to a biotinylated phalloidin molecule or actin protein. Taken together, this body of work represents strategies for the labeling and visualizing, both freely diffusing and actin tethered, long- RNAs and their interactome in real-time. xii Chapter I Under the Microscope: A Historical Overview of Single-Molecule Approaches and Analyses of Intracellular RNA1 1.1 INTRODUCTION The eukaryotic cell is highly complex.