Investigating the competing endogenous RNA hypothesis Genome-wide and in Single Cells by Apratim Sahay B.S in Physics and Mathematics, University of Chicago (2008) Submitted to the Department of Physics I- in partial fulfillment of the requirements for the degree of CO cO- C DOCTOR OF PHILOSOPHY at the I Ul) MASSACHUSETTS INSTITUTE OF TECHNOLOGY June 2015 Massachusetts Institute of Technology 2015. All rights reserved. Signature redacted Author. Department of Physics /1 May 22nd, 2015 Signature redacted Certified by A/ /7 Alexander van Oudenaarden MIT Pro sor of Physics and Professor of Biology Director, Hubrecht Intitute for evelopmental Biology I Thesis Supervisor Certified by Signature redacted Jeff Gore Latham Family Career Development Assistant Professor of Physics Thesis Supervisor Signature redacted_ _ Accepted by Professor Nergis Mavalvala Associate Department Head of Physics 77 Massachusetts Avenue Cambridge, MA 02139 MITLibraries htp://Iibraries.mit.edu/ask DISCLAIMER NOTICE Due to the condition of the original material, there are unavoidable flaws in this reproduction. We have made every effort possible to provide you with the best copy available. Thank you. The images contained in this document are of the best quality available. Investigating the competing endogenous RNA hypothesis Genome-wide and in Single Cells by Apratim Sahay Submitted to the Department of Physics on May 22nd, 2015, in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Abstract The observation that microRNAs (miRNAs), through a titration mechanism can cou- ple interactions of their common targets (competing endogenous RNAs or ceRNAs) has prompted a general "ceRNA hypothesis' that RNAs can regulate each other indirectly through global RNA-miRNA-RNA networks. These ceRNAs are said to "crosstalk' with each other by competing for common miRNAs. Although many individual ceRNAs have been found, fundamental questions about both the magnitude and generality of the crosstalk effect remain. In our study we combine RNA sequencing and single-molecule FISH (smFISH) approaches to both measure the magnitude of the crosstalk effect genome-wide by perturb- ing three known ceRNAs (Pten, Vapa, Cnot6l) and to identify mechanisms by which the crosstalk effect acts. We identify hundreds of putative ceRNAs and dissect the contributions of individual miRNAs in transmitting crosstalk. We demonstrate that while the crosstalk effect is pervasive, it nevertheless remains bounded by the size of the perturbation. Further- more, we show that both the number and affinity of shared miRNA binding sites between targets is crucial in determining the magnitude of the crosstalk strength. Using the smFISH data, we examined the single-cell gene expression profiles of pairs of ceRNAs and found that ceRNA gene expression is correlated only in the presence of active miRNAs. Additionally, on inspecting the intra-cellular localization of RNA molecules, we found a miRNA-dependent colocalization of ceRNAs, suggesting a new signature of crosstalk between ceRNAs that extends and modifies the original hypothesis. Thesis Supervisor: Alexander van Oudenaarden Title: MIT Professor of Physics and Professor of Biology Director, Hubrecht Institute for Developmental Biology Thesis Supervisor: Jeff Gore Title: Latham Family Career Development Assistant Professor of Physics This work is dedicated to my grandparents Gaur Priya Devi & Krishnanand Sahay, Veena Srivastava & Shailendra Nath Srivastava who instilled in me their love for the life of the mind and the desire to share its fruits with others. Acknowledgements This thesis would not have been possible without the help, encouragement and support of many people to whom I owe a debt of gratitude. First and foremost, Alexander van Oude- naarden, my thesis advisor, who welcomed me into his lab and gave me great freedom and support throughout my PhD. Alexander's grasp of experimental biophysics is truly broad and deep, which I found as he led the lab through the smFISH era, the RNA sequencing era and the single-cell sequencing era. Not only was he an inspiring scientist, but he also created a fantastic group of enormously talented students and post-docs in building 68 that buzzed with stimulating ideas. After introducing me to microRNAs and suggesting an ex- perimental plan of attack, he then stepped back to let me find my own way. Always there to offer a suggestion, to share in excitement or to help think through a problem, he has been a great mentor. After his move to Utrecht, he offered me numerous opportunities to visit him there and work with another set of fantastic people. Finally, I am also thankful for the opportunity as a graduate student to be able to make mistakes. I will be forever grateful for Alexanders limitless patience throughout this process. I sincerely thank my thesis committee members, Jeff Gore, Jeremy England and Mehran Kardar for their support and advice throughout my graduate years. Jeff in particular for his blend of unflappable enthusiasm and guidance during some of the more trying phases of research. Next, my wonderful collaborators - Joern Schmiedel, Yannan Zheng, Sandy Klemm, Dominic Gruen. Joern came to MIT a year into my thesis project and has helped shape and sharpen my ideas tremendously. His enthusiasm and dogged persistence in solving problems were a great boost whenever I was stuck in dark alleys. Yannan and I started vi and finished our PhD's together and also been through all the ups and downs of graduate student life together. She taught me a lot about microRNA biology and was an invaluable source of experimental guidance, especially cell culturing and cloning. Dominic helped set up the RNA Sequencing pipeline in Utrecht and generously shared his expertise in microRNA bioinformatic analysis. Sandy was a fantastic friend, a critical sounding board for hypothesis, and taught me the intricacies of live-cell FACS sorting. My graduate life would not have been half as much fun without the tremendous people at the AvO lab: Dong Hyun Kim for mentoring me in worm biology when I first came to the lab and training me in the dark arts of FISH. He and Christoph Engert were vital founts of friendship, mentorship and cheer. To the postdocs: Stefan, Jeroen, Magda, Nick,Nikolai, Lenny, Shalev, Philipp, Anna, Gregor, Arjun, Scott, who took the time to provide critical advice on experiments, research, and life. To the amazing graduate students in the lab office who shared all the joy and frustrations of research. You made the AvO lab fun and exciting: Ruizhen, Miaoqing, Bernardo, Clinton, Ni, Annnalisa, Dylan, Kay, Juan, Shankar, Hyun. Lastly, Monica Wolf, Annemiek van Rooijen, Crystal, Cathy and Katie who have meticulously taken care of any and all administrative issues that have cropped up. During my time at MIT, I've been lucky to have some wonderful roommates and friends- Michelle, Andrew, Andrew Stecker, Arghavan, David who have been fantastic at keeping a balanced life. Friends on the squash courts who have offered huge support and camaraderie over the years, thank you for helping me maintain my sanity- Najib, Ann, Pam, Jan, Frans, Christopher, Christoph, Justin, Mehmood. Finally I would like to thank my parents Aparajita and Avinash, for being so amaz- ingly supportive throughout my entire academic career, and life in general, and providing countless opportunities to me. My sisters Ananya and Apoorva for your love and feigned excitement at my research! My cousins, Sunny, Pranay, and Abhilash for their encourage- ment and shared geekdom. My extended family in India for their tremendous support over the years. Lastly, my wife Liz, without whom I would never have been introduced to the world of biology, and without whose unwavering support none of this would have happened. Your intelligence, encouragement and limitless love makes all things possible. vii Table Of Contents Acknowledgements vi List of Figures xii 1 Introduction 9 1.1 MicroRNAs-discovery, biogenesis, target binding and competition . 10 1.1.1 Discovery of miRNA Regulation ..... .... ..... .... 10 1.1.2 Biogenesis of miRNAs ........ .... ............. 11 1.1.3 miRNAs: target binding and competition . ............. 12 1.2 ceRNAs: Discovery .................. ... .... .... .. 13 1.2.1 Different types of endogenous ceRNAs . .. ... .... .... .. 15 1.2.2 3'UTRs as ceRNAs ............. .. .... .... ... 15 1.2.3 Circular RNAs ................ .. .... .... ... 16 1.2.4 Pseudogenes as ceRNAs ........... .... ..... ... 16 1.2.5 Long non coding (lncRNA) as ceRNAs ... ......... ... 17 1.3 Modulators of crosstalk activity ........... .... ..... .... 18 1.3.1 Abundance of miRNA binding sites and miRNA concentration .. 19 1.3.2 MiRNA binding affinity ....... ............ ..... 20 1.3.3 MRE Accessibility and Local concentrations ............. 20 1.3.4 Post-transcriptional network effects ...... ............ 21 1.4 Summary and Outline ...................... ....... 22 2 Assesment of the ceRNA hypothesis with integrated genome-wide mea- surements reveals bounded yet pervasive crosstalk activity 24 2.1 Results .. ......... ........ ......... ........ .. 26 2.1.1 ODE biochemical model of crosstalk predicts that crosstalk strength should be bounded by 1 .................. ...... 28 2.1.2 Quantification of crosstalk following siRNA knockdown of sender 32 2.1.3 Pervasive yet bounded mRNA Crosstalk upon siRNA knockdown. 35 2.1.4 Crosstalk strength correlates with the number of shared binding sites 37 2.1.5 miRNA's hierarchically contribute to transmitting