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Genome-wide Investigation of Cellular Functions for tRNA Nucleus- Cytoplasm Trafficking in the Yeast Saccharomyces cerevisiae DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Hui-Yi Chu Graduate Program in Molecular, Cellular and Developmental Biology The Ohio State University 2012 Dissertation Committee: Anita K. Hopper, Advisor Stephen Osmani Kurt Fredrick Jane Jackman Copyright by Hui-Yi Chu 2012 Abstract In eukaryotic cells tRNAs are transcribed in the nucleus and exported to the cytoplasm for their essential role in protein synthesis. This export event was thought to be unidirectional. Surprisingly, several lines of evidence showed that mature cytoplasmic tRNAs shuttle between nucleus and cytoplasm and their distribution is nutrient-dependent. This newly discovered tRNA retrograde process is conserved from yeast to vertebrates. Although how exactly the tRNA nuclear-cytoplasmic trafficking is regulated is still under investigation, previous studies identified several transporters involved in tRNA subcellular dynamics. At least three members of the β-importin family function in tRNA nuclear-cytoplasmic intracellular movement: (1) Los1 functions in both the tRNA primary export and re-export processes; (2) Mtr10, directly or indirectly, is responsible for the constitutive retrograde import of cytoplasmic tRNA to the nucleus; (3) Msn5 functions solely in the re-export process. In this thesis I focus on the physiological role(s) of the tRNA nuclear retrograde pathway. One possibility is that nuclear accumulation of cytoplasmic tRNA serves to modulate translation of particular transcripts. To test this hypothesis, I compared expression profiles from non-translating mRNAs and polyribosome-bound translating mRNAs collected from msn5Δ and mtr10Δ mutants and wild-type cells, in fed or acute amino acid starvation conditions. Microarray data revealed that several amino acid biosynthetic pathways, including the sulfur assimilation, arginine ii biosynthesis, and leucine biosynthesis pathways, are primary targets of the tRNA trafficking processes. I confirmed the microarray data by both Northern and Western blot analyses. Levels of all tested target proteins involved in such amino acid biogenesis pathways are down-regulated when the tRNA nuclear import or re-export is disrupted. The steady state levels of target total RNAs are similar between wild-type cells and tRNA trafficking defective mutants. The data suggest that the reduction of target proteins most likely results from translation defects of the target mRNAs. This study provides information that tRNA nuclear-cytoplasmic dynamics is connected to amino acid biosynthesis via control at the level of translation. iii This thesis is dedicated to my family and the memory of my dear mother. iv Acknowledgements First and foremost I want to thank my advisor Anita K. Hopper, for her warm encouragement and advice. From her I have learned how to enjoy the research life and open my mind for every possibility. She is always patient, optimistic, and enthusiastic, even during difficult moments. I appreciate all her supports and contributions of time, ideas, and funding in my Ph.D pursuits. When I lose my confidence, she always believed me more than myself. I thank her wide knowledge and logical way of thinking, which made this thesis possible. I am grateful for the excellent role model which she provided as a woman scientist. It is my honor to be one of her students. I am acknowledged my thesis committee, Professor Stephen Osmani, Professor Kurt Fredrick, and Professor Jane Jackman, for their interests, professional comments, and personal encouragements. I am also grateful that my previous advisor in the Nationwide Children Hospital, Dr. Akihira Otoshi, for his giving me the chance coming to the United States. I want to thank the director of MCDB graduate program, Professor David Bisaro, for his understanding and decision of accepting me, which made me get the opportunity to join in this wonderful lab. Being part of the Hopper lab would definitely become a beautiful memory in my life. It was my pleasure to work with such nice lab members. I thank our post-docs Rebecca, for her good ideas and always willing to help us whenever we have questions, v and Emily, for her nice communication skills. Thanks to Greetchen, Nripesh, Tsung-Po, Hsiao-Yun (Ivy), Jingyan, and Marina. When I became too serious or too panic, they provided laughs and supports. When I had successful experiments, they shared the joyful moments. My life in the OSU was fulfilling and the learning experience was most comprehensive in my study journey. As a hearing impaired foreigner, I sincerely thank Cindy Knecht and Andrea Crago from the disability service that was provided by the OSU and the Professional Reporters, Inc. Their amazing real-time transcribing work truly helped me overcome the double barriers of disability and language. Thank to OSU provided such a friendly campus. I thank people in the department of Molecular Genetics, in particular Professor Jim Hopper, Professor Paul Herman, and their lab members. In these years of joys and frustrations, thanks for all the supports my friends in Columbus. Thanks to Rosa, Keith and their dog Bailey, for their generosity and warm supports. Thanks to I-Ju, Liang-Chun, and Yuh-Ying, for banding together over food and life. I also thank Kuo-Fang and Shu-Hao, studying together with them was a different and great experience. Also thanks to many old friends in Taiwan, they keep caring about me no matter the distance. Finally, and most importantly, thanks to my parents for their nurture and education. Their unconditional love made me brave to face the challenges and was the motivation of my pursuits. Thanks to my sister and brother, and their families, for their understanding and supports. And last, but not least, to Yuan-Sheng, thank you for the love and consistent being there for me. vi Vita March 1978 ....................................................Born−Taipei, Taiwan 2000...............................................................B.S. Biology, National Taiwan Normal University, Taiwan 2003................................................................M.S. Genetics, National Yang-Ming University, Taiwan 2006 to present ..............................................Graduate Research Associate, Molecular, Cellular, and Developmental Biology Program, The Ohio State University Publication Lai, Y.-S., Murali, S., Ju, H. Y., Wu, M. F., Guo, I. C., Chen, S. C., Fang, K. and Chang, C. Y. Two iridovirus-susceptible cell lines established for kidney and liver of grouper, Epinephelus awoara, and partial characterization of grouper iridovirus. Journal of fish disease 23, 379-338, 2000. vii Lai, Y.-S., Murali, S., Chiu, H. C., Ju, H. Y., Lin, Y. S., Chen, S. C., Guo, I. C., Fang, K. and Chang, C. Y. Preparation of yellow grouper nervous necrosis virus in a new nodavirus- susceptible cell lines from yellow grouper, Epinephelus awoara brain tissue. Journal of fish disease 24,299-309, 2001. Chu, H. Y. Phenotypic Analysis of the Aurora-A Transgenic Mice. Master Thesis. Institute of Genetics, National Yang Ming University, July, 2003. Chu, H. Y. and Otoshi, A. Cloning and functional analysis of hypothalamic homeobox gene Bsx1a and its isoform, Bsx1b., Mol Cell Biol. 27(10), 3743-9, 2007. Li, C.C., Chu, H.Y., Yang, C.W., Chou, C.K. and Tsai, T.F. Aurora-A overexpression in mouse liver causes p53-dependent premitotic arrest during liver regeneration. Mol Cancer Res. 7(5), 678-88, 2009. Chu, H. Y. and Hopper, A. K. Genome-wide investigation of the cellular functions for tRNA nucleus-cytoplasm trafficking in S. cerevisiae. (in prep.) Fields of Study Major Field: Molecular, Cellular and Developmental Biology viii Table of Contents Abstract ............................................................................................................................... ii Acknowledgements ............................................................................................................. v Vita .................................................................................................................................... vii Fields of Study ................................................................................................................. viii Table of Contents ............................................................................................................... ix List of Tables ................................................................................................................... xiv List of Figures .................................................................................................................. xvi Chapter 1 Introduction.................................................................................................. 1 1.1 Nucleocytoplasmic transport ................................................................................ 1 1.2 tRNA biology in yeast .......................................................................................... 5 1.2.1 The life of tRNA: biogenesis, maturation, and turnover ........................... 5 1.2.2 tRNA subcellular dynamics ..................................................................... 10 1.3 Amino acid stress responses ............................................................................... 12 1.4 Microarray analysis ...........................................................................................
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