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CHAPTER 1 Translational Reprogramming in Cellular Stress Response

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CHAPTER 1 Translational Reprogramming in Cellular Stress Response INVESTIGATING TRANSLATIONAL REPROGRAMMING IN CELLULAR STRESS RESPONSE BY ELONGATION PAUSING AND ALTERNATIVE INITIATION A Dissertation Presented to the Faculty of the Graduate School of Cornell University In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy by Botao Liu May 2014 © 2014 Botao Liu INVESTIGATING TRANSLATIONAL REPROGRAMMING IN CELLULAR STRESS RESPONSE BY ELONGATION PAUSING AND ALTERNATIVE INITIATION Botao Liu, Ph.D. Cornell University 2014 Cell survival in changing environments requires appropriate regulation of gene expression, including translational control. Multiple stress signaling pathways converge on several key translation factors and rapidly modulate mRNA translation at both the initiation and the elongation stages. Here, I discover that intracellular proteotoxic stress reduces global protein synthesis by halting ribosomes on transcripts during elongation. Deep sequencing of ribosome-protected mRNA fragments reveals an early elongation pausing, roughly at the site where nascent polypeptide chains emerge from the ribosomal exit tunnel. Inhibiting endogenous chaperone molecules by a dominant-negative mutant or chemical inhibitors recapitulates the early elongation pausing, suggesting a dual role of molecular chaperones in facilitating polypeptide elongation and co-translational folding. My results further support that trapped chaperone under stress may prevent the release of elongation factors from ribosomes. My study reveals that translating ribosomes fine-tune the elongation rate by sensing the intracellular folding environment. The early elongation pausing represents a co-translational stress response to maintain the intracellular protein homeostasis. Correspondingly, repression of global protein synthesis is often accompanied with selective translation of mRNAs encoding proteins that are vital for cell survival and stress recovery. Understanding the selective translational control in gene expression relies on precise and comprehensive determination of translation initiation sites (TIS) across the entire transcriptome. Here, I develop an approach (global translation initiation sequencing, GTI-seq) to achieve simultaneous detection of both initiation and elongation events on a genome-wide scale. With single nucleotide resolution, I show an unprecedented view of alternative translation initiation in mammalian cells. Furthermore, I uncover a robust translational reprogramming of protein catabolic process, in particular the proteasome system, in response to starvation. This regulatory mode of TIS selection indicates that the scope of selective translation under stress conditions is much broader than anticipated. Collectively, my studies have revealed unprecedented proteome complexity and flexibility through stress-induced translational reprogramming, including ribosome pausing during elongation and wide-spread alternative translation initiation. Elucidation of the regulatory mechanisms underlying translational reprogramming will ultimately lead to the development of novel therapeutic strategies for human diseases. BIOGRAPHICAL SKETCH Botao Liu was born December 4, 1987 in Ganzhou, Jiangxi, China. He earned his Bachelor of Science from Zhejiang University in China 2009. During his undergraduate studies, he received numerous honors and awards, including Excellent Graduates with Honors, Outstanding Thesis for Bachelor's Degree, National Scholarship of China, and Excellent Undergraduate Scholarship. He also served as an undergraduate teaching assistant in 2007-2008 and the Director of Academic Department, Student Union, College of Life Sciences in 2006. He travelled to United States first in 2008 during a summer research program at North Carolina State University. In 2009, Botao Liu joined the doctoral program of Genetics, Genomics and Development at Cornell University. He was awarded the Hsien Wu and Daisy Yen Wu Scholarship/The Liu Memorial Award and Genomics Scholars Award in 2013. He served as a teaching assistant for the Department of Molecular Biology and Genetics in 2013 and a research assistant for the Division of Nutritional Sciences from 2010 to 2014. Outside of academia, Botao Liu interned at the Department of Ion Torrent, Thermo Fisher Scientific Inc in 2013. Botao Liu presented his research in Molecular Chaperones & Stress Responses Meeting at Cold Spring Harbor in 2012 and published six research and review papers in high-profile journals. Additionally, he is also one of three inventors of a US patent derived from his work. After earning the Ph.D., he plans to continue with a period of postdoctoral training and then look for a faculty position in major institutions or a research and development job in the biotechnology and pharmaceutical industry. iii I delicate my dissertation work to my parents, Huazhou Liu and Lanfen Gu, who have given me their unreserved love, support, and trust. 献给我的父母 iv ACKNOWLEDGMENTS First, I would like to express sincere appreciation to my supervisor, Dr. Shu- Bing Qian, for all the time and effects that he has put into my graduate studies in the past five years. He has supported me both scientifically and personally. He is very approachable as a supervisor and I still remember all the inspirational discussions during late nights and weekends. His suggestions are always insightful and extremely helpful, which guide me to the effective path. He is open to my crazy ideas and risky proposals at all times. When new discoveries emerge, his excitement and enthusiasm about science encourage and inspire me. When inevitable difficulties occur, his calmness, patience, and persistence keep me away from anxiety and frustration. In addition, he has spent tremendous amount of time on improving my scientific writing and presentation skills. Finally, he indeed cares about the career of students and allows me to explore possibilities in both academia and industry. I would never have been able to finish my dissertation without the guidance from Shu-Bing. He has fully practiced his philosophy of advisor and student relationship - equality, dialogue, communication and understanding. I would also like to thank Dr. Scott Emr and Dr. Richard Cerione for their research expertise and insightful advice. In my annual committee meetings, I always benefit a lot from their questions, critiques, and suggestions to my project. Their unique perspectives have led me to think about my research in a bigger picture of biology. Scott and Richard have also become role models for me and they have shown me that researchers can be both successful scientists and helpful educators. v In addition to my special committee members, I also appreciate other members of MBG department and scientific community in general for providing a nourishing environment. Specifically, I am thankful for Dr. Dan Barbash, who appreciated my research potential and let me join GG&D field despite of the unsatisfactory GRE and TOEFL scores. I‟m also grateful to Drs. Eric Alani, John Parker, Paul Soloway, Jun (Kelly) Liu, Siu Sylvia Lee for their tremendous help and Vicki Shaff for her support throughout my studies. I would also thank all the anonymous reviewers who have read and criticized my studies. Although the review process is actually lengthy and painful, I have learned from peer scientists to be cautious and responsible to the results, interpretations and conclusions. I feel extremely lucky of spending such a wonderful time with all the former and current Qian lab members. Special thanks go to Dr. Yan Han. It was Yan who convinced me to join Qian lab after my rotations. More importantly, I learned most of the experimental skills used throughout my graduate studies from her, from the basic tissue culture to the cutting-edge ribosome profiling technology. She is also my close friend outside of lab and I consider her as a lovely sister who has warmed my heart in this cold town far away from home. I thank former graduate student Dr. Crystal Conn, who trained me during my rotation and accompanied me during all the nights and weekends in the lab. Her optimism, energetic personality, and kindness have been encouraging me all the time. I am grateful to Drs. XingQian (Ben) Zhang and Xiangwei Gao for all the valuable discussions about science and their help in many experiments. I also appreciate the wonderful and productive collaboration with Drs. Sooncheol Lee and Soohyun Lee, which led to most of the results in Chapter 3. I also vi have enjoyed all the scientific and social debates with R. Alex Coots, who has provided me with a broader view of the culture, society and scientific community of US. Additionally, I still miss all the happiness shared with Dr. Jun Sun and Xiaoxing (Alva) Shen. Finally, I really appreciate new perspectives, novel thoughts, and supports brought by recent Qian lab members, including Rada Omanovic, Julianna Magdalon, Drs. Ji Wan, Jun Zhou, and Mridusmita Saikia. I would never have been able to persist in my adventure of science without the support from all my friends over the world. As classmates in college, Xiang Ma, Zhisong He, Yixing Zhai, Song Feng and I may be far from each other geographically now, but we are close in the journey of pursuing science. We encourage each other in face of difficulties and share the excitement about breakthroughs. We exchange opinions on scientific problems from distinct perspectives. In addition, I would like to specially thank my friends at Cornell, including Lu Huang, Tao Sun, Donghao Li, Mingfei Ji, Xian Qu, Yao Sun, Dr. Zhilong Bao, Dr. Zhen
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