STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF THE RNA EXOSOME FROM S. CEREVISIAE by Elizabeth V. Wasmuth A Dissertation Presented to the Faculty of the Louis V. Gerstner, Jr. Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy New York, NY July, 2015 _________________________________________ __________________ Christopher D. Lima, PhD Date Dissertation Mentor Copyright by Elizabeth V. Wasmuth 2015 © DEDICATION This work is dedicated to those young people who look past the fences and see the vast landscape beyond, who do not see the boundaries and their limitations but see opportunities for growth and adventure. And in memory of M.R. Wasmuth for her example of perseverance, dignity, and selflessness. iii ABSTRACT The RNA exosome is an essential, multisubunit complex found in eukaryotes that houses 3’ to 5’ exoribonuclease and endoribonuclease activities. It is a major player in a diverse set of processes, including, but not limited to, normal RNA turnover, quality control, biogenesis and maturation pathways in the cytoplasm, nucleus, and nucleolus. Its 9 subunit non-catalytic core (Exo9) is composed of a two-stacked ring, featuring a prominent central channel. Two enzymes, Rrp44 and Rrp6, associate with Exo9, both possessing 3’ to 5’ exoribonuclease activity. The exosome was first discovered and its constituents described in 1997 – soon after, it became clear that different compositions of exosomes exist in various cellular compartments – for example, in the cytoplasm of S. cerevisiae, Rrp44 associates with Exo9, while Exo9, Rrp44 and Rrp6 form the nuclear exosome. It has been thought that various flavors of exosomes exist to allow targeting of distinct RNA substrates, but how a common Exo9 core could impart very different activities to its associated ribonucleases had not been well understood. Chapter 1 of this dissertation begins by describing the findings of biochemical and genetic studies that probed the functions of the exosome, namely that Exo9 regulates both Rrp44 and Rrp6, that Rrp6 significantly activates Rrp44 in the 11 subunit nuclear exosome, and that all three enzymatic activities rely on the integrity of the Exo9 central channel. Chapter 2 then focuses on addressing questions regarding Rrp6-associated exosomes. Specifically, 1) how does Rrp6 bind to the Exo9 core, 2) how does the Rrp6 exosome engage RNA (or any member of bacterial RNase D for that matter), and 3) how does the Rrp6 activation of Rrp44 in the nuclear exosome occur? Discussion will be centered on insights gained from the crystal structure of a Rrp6-Exo9 exosome engaged with polyA RNA, a decay substrate in the nucleus, and on subsequent structure-function analysis. Briefly, the highly conserved catalytic module of Rrp6 rests atop the Exo9 core iv and central channel on one face of the S1/KH ring, with its non-catalytic C-terminus tethering Rrp6 to the core. The 3’ end of the RNA is engaged in the Rrp6 active site, then threads into the S1/KH ring of the Exo9 core. With both Rrp44 and Rrp6 active sites positioned on opposite ends of the Exo9 central channel and using partially overlapping yet distinct paths for RNA decay, it is now appreciated that the basis for 3’ to 5’ directionality rests solely on Rrp6 and Rrp44 active sites, with Exo9 bearing no specificity for 3’ ends. The final two chapters will focus on the large contribution of small, disordered regions to greater exosome function – a polypeptide stretch dubbed the Rrp6 “lasso” in Chapter 3, and a basic protein cofactor, Mpp6, in Chapter 4. Using a combination of biochemical and genetic techniques, the Rrp6 lasso has been found to have an integral role in recruiting RNA towards the exosome, directing its path of ingress, and in facilitating decay by all three ribonuclease activities of the exosome. Mpp6, meanwhile, primarily modulates Rrp6 activity in an exosome-dependent fashion. Biochemical and preliminary structural results will be discussed to elucidate how Mpp6 stimulates exosome decay. v ACKNOWLEDGMENTS Harold Varmus, a former president of Sloan Kettering, once mentioned the importance of a gate keeper in shaping the future of a young person. In his case, the gate keeper in his life was a former science mentor whose example inspired the young Varmus to change his career path, turning him on to experimental science, asking the “hows” and the “whys” rather than the “whats” of life. This story resonated with me because I did not grow up wanting to be a scientist – on the contrary, it was a paleontologist digging the bones of extinct animals, and then a veterinarian fixing the bones of living animals. I had no desire to question existing protocol. I was happy to read textbooks, accepting the dogma of whatever it was I read. This has radically changed. Here I wish to acknowledge the gate keepers in my life, who have positively impacted me and inspired me to do the same for others. In all cases, their influence extends beyond just science, and for this, I am forever grateful. I first wish to thank my parents, Scott and Victorina, and sisters Abigail and Christine, for their infinite support and advice in life decisions, and for grounding me with good values. To my grandparents William and Norma, for their progressiveness, and examples in juggling politics in academics while maintaining integrity. To my high school English teacher, Don Delo, who angered and challenged me by his then accurate statement that I am a “black and white” thinker. I’d like to think he’d be pleased with my current color palette. To my physician-scientist friend, Shahrukh Ali, whose love for science first eluded me from high school through college, but whose advice to do a summer undergraduate research fellowship in the lab of crystallographer, Dr. Mair Churchill at UC Denver changed my career path. To Mair Churchill, and my mentor, Doug Donham, who gave a very green undergraduate a summer in their lab, despite constraints on their time, budget and vi patience. During this formative time, I was shaken by the power of science, specifically structural biology, to explain mechanistic concepts I blandly swallowed in undergraduate biochemistry. Furthermore, I learned I could find these answers with my own hands; results – both positive and negative – were gratifying. To Xingen Lei at Cornell University, who appointed me as a technician in his lab, giving me an unprecedented amount of responsibility and trust in managing a project, and fantastic mentorship and great appreciation. To Forbes Porter and Christopher Wassif, whose genetics lab I worked at the NIH. From Chris I learned to think about the end goal of an experiment, and how to disassociate the negative results of science from life. To Sasha Serganov, with whom I was privileged to work, who encouraged me to question the actual setup of failed experiments, not solely myself. To my committee member, Dinshaw Patel, for his support and advocacy throughout the years, and his ability to challenge me by inquiring about the status of my project, and noting the possibility of doing the “impossible” when the Conti lab published the first yeast exosome structure. To my committee member, Scott Keeney, for his mentorship from day one, and his dedication to strengthening my character and scientific abilities. Were it not for him, I would not be where I am now. To Elena Conti, academically a competitor, but personally a mentor and source of inspiration. I am grateful for her advice, and her willingness to provide it, throughout the years. To the members of the Lima lab, both past and present for their collegiality and friendships. The lab has been an ideal environment to work and grow mainly because of the wonderful people in it. I’d like to highlight my baymates, Agni Ghosh and Laurent Cappadocia for their constant advice, and for serving as role models in providing vii mentorship in an infinitely patient manner. Shaun Olsen for his friendship, scientific and career support, and unconventionality. Nancy Arango, Chuck Streich, Xavier Mascle, Zac Hann and Selom Doamekpor for their help. And the exosome team: current members John Zinder and Kurt Januszyk, and previous members Jaclyn Greimann and Victor Liu. Finally to Christopher Lima, for taking a gamble on a non-specialized, maverick graduate student, and entrusting her with his extremely challenging, competitive and personal project. My scientific and personal growth has been immense these years because of the environment he has provided me. It was a privilege and honor to work with him in the trenches during the exciting moments of discovery earlier on - these times are some of the fondest in my life. I also thank him for later giving me extreme independence. I am confident that I am leaving his lab a better scientist, thinker, and person, and look forward to tackling the challenges that lie ahead. viii TABLE OF CONTENTS LIST OF TABLES ............................................................................................................. xii LIST OF FIGURES ......................................................................................................... xiii INTRODUCTION .............................................................................................................. 1 Discovery of the RNA exosome .................................................................................... 3 Global architecture of the eukaryotic exosome core ..................................................... 4