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Transcript-Specific Cytoplasmic Degradation of YRA1 Pre-Mrna Mediated by the Yeast EDC3 Protein: a Dissertation

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Transcript-Specific Cytoplasmic Degradation of YRA1 Pre-Mrna Mediated by the Yeast EDC3 Protein: a Dissertation University of Massachusetts Medical School eScholarship@UMMS GSBS Dissertations and Theses Graduate School of Biomedical Sciences 2007-12-17 Transcript-Specific Cytoplasmic Degradation of YRA1 Pre-mRNA Mediated by the Yeast EDC3 Protein: A Dissertation Shuyun Dong University of Massachusetts Medical School Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/gsbs_diss Part of the Amino Acids, Peptides, and Proteins Commons, Biochemistry Commons, Bioinformatics Commons, Cells Commons, Computational Biology Commons, Fungi Commons, Genetic Phenomena Commons, Genetics Commons, Genomics Commons, Molecular Biology Commons, Molecular Genetics Commons, and the Nucleic Acids, Nucleotides, and Nucleosides Commons Repository Citation Dong S. (2007). Transcript-Specific Cytoplasmic Degradation of YRA1 Pre-mRNA Mediated by the Yeast EDC3 Protein: A Dissertation. GSBS Dissertations and Theses. https://doi.org/10.13028/ckcv-mt21. Retrieved from https://escholarship.umassmed.edu/gsbs_diss/352 This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in GSBS Dissertations and Theses by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. A Dissertation Presented By Shuyun Dong Submitted to the Faculty of the University of Massachusetts Graduate School of Biomedical School, Worcester In partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY IN BIOMEDICAL SCIENCE December 17, 2007 TRANSCRIPT-SPECIFIC CYTOPLASMIC DEGRADATION OF YRA1 PRE-mRNA MEDIATED BY THE YEAST EDC3 PROTEIN A Dissertation Presented By Shuyun Dong The signatures of the Dissertation Defense Committee Signifies completion and approval as to style and content of the Dissertation ____________________________________________ Allan Jacobson, Ph.D., Thesis Advisor ____________________________________________ Craig Peterson, Ph.D., Member of Committee ____________________________________________ Peter Pryciak, Ph.D., Member of Committee ____________________________________________ Phillip Zamore, Ph.D., Member of Committee ____________________________________________ Robert H Singer, Ph.D., Member of Committee The signature of the Chair of the Committee signifies that the written dissertation meets the requirements of the Dissertation Committee ____________________________________________ Richard Baker, Ph.D., Chair of Committee The signature of the Dean of the Graduate School of Biomedical Sciences signifies that the student has met all graduation requirements of the school ____________________________________________ Anthony Carruthers, Ph.D., Dean of the Graduate School of Biomedical Sciences Department of Molecular Genetics and Microbiology December 17, 2007 ii COPYRIGHT Chapter II of this thesis has been published as: Dong, S., Li, C., Zenklusen, D., Singer, R., Jacobson, A., and He, F. (2007) YRA1 autoregulation requires nuclear export and cytoplasmic Edc3p-mediated degradation of its pre-mRNA. Molecular Cell. 25(4): 559-573. iii ACKNOWLEDGEMENTS I would like to dedicate this dissertation to my beloved father who passed away while I was pursuing my Ph.D. degree. I am deeply indebted to my wonderful, wonderful parents for their support and sacrifices for my decision to be away from them and pursue my doctoral degree in the United States of America. I also deeply thank my brother for his quiet unselfish devotion. Since I left my hometown twenty years ago, he has taken all the family responsibility and never complains to me. I am truly grateful to my advisor, Dr. Allan Jacobson. Having worked with him in the past six and half years has been an extraordinary experience to me. I thank Dr. Jacobson for providing outstanding research opportunities and complete support from financial, laboratory, and intellectual perspectives. With his unique patience, persistence, and intelligence, Dr. Jacobson has made my graduate study such an exciting and rewarding one. There are countless stories which are worthy of my forever remembrance and appreciation. I can never forget those days when he watched my presentation rehearsal again and again. I also always remember his great patience of helping me to improve my English writing. To Dr. Jacobson, I accredit my all successes. I will be working hard in the future so that he will be proud of having me as his student. Thank you, Dr. Jacobson. iv Another indispensable guider throughout my Ph.D. study is Dr. Feng He. I received his help since the first day I joined Dr. Jacobson lab. I am always motivated by his passion to science. Whenever I encountered difficulties in my work, I would like to talk to him. I know I can always find both intelligent and spiritual support from him. He is always available not only when I need help in scientific research but also when I have life difficulties. Without him, I can not complete such a dissertation. My sincere thanks also go to the members of my thesis committee, Dr. Richard Baker, Dr. Craig Peterson, Dr. Peter Pryciak, Dr. Phillip Zamore, and Dr. Robert H Singer. Each of them has made a lot of positive influence on my academic advancement and has taken extra time to help me. I thank Dr. Baker for serving as the chair of the committee and in charge of a lot of paper work for me. From him, I also learned how to clearly and effectively present my ideals as a scientist. I thank Dr. Peterson for his inspiration, enthusiasm, guidance and support during my graduate study. I thank Dr. Pryciak for his critical comments on my research and generous assistance in so many years as a member of my qualification committee, thesis committee, and defense committee. I appreciate Dr. Zamore for his commitment to my study and his guidance as I prepare for the next stage of my career. I am especially grateful to Dr. Robert H Singer for his scientific help and collaboration, which have fundamentally shaped my work. v Dr. Jacobson lab is such a wonderful group. People here are smart, lovely and supportive. I thank Dr. Marcus Jonhansson for his continuous encouragement, advice, and the happy tennis time which help me to build a healthy body. I thank Mr. Shubhendu Ghosh for helping me learn the polyribosome profile analysis and helping me to accommodate the life in US as a foreigner. I thank Dr. David Mangus for generous sharing plasmids and reagents and helps in my work. I thank Dr. Nadia Amrani for the thoughts and helps in the work. I thank Dr. Stephanie Kervestin for teaching me how to do Western blotting analysis. I thank Ms. Phyllis Spatrick for helping me to finish so many microarray analyses. I thank Ms. Robin Ganesan for generous sharing plasmid and yeast strains. I thank Ms. Chunfang Li for being a good friend of me in the lab. I would like to give a very special thank to Ms. Debra Celularo for ordering reagents and preparing a lot of paper work for me. I appreciate Dr. Daniel Zenclusen who helped me to finish the FISH experiment in my thesis. I also thank the great helps from the graduate student office and the international office. I am grateful to the assistance of Ms. Gaile Arouette, Ms. Michelle Deignan, and Ms. Barbara LeFebvre. I am so fortunate to be able to accomplish this challenging work also because I made a lot of friends in America. They are Dr. Daniel Young, Mr. Xiaochu Cheng, Mr. Lue Dai, Ms. Tingting Du, Mrs. Yanxia Guo, Dr. Yuqing Hou, Ms. Lixia Li, Dr, Su-chen Lin, Ms. Min Shi, Dr. Xiaoting Wang, Dr. vi Yuanfei Wu, Ms. Fenhui Xu, Dr. Xiaofang Yang, Mr. Lijian Yu. I also received unforgettable support from many of my relatives and friends in China. I especially thank Dr. Chengzhang Chen,Dr. Shengzhang Dong, Dr. Zhenxin Zhao, Dr. Saixian Zhong, Dr. Daming Li, Dr. Hong Song, Dr. Qiansheng Hu, Dr. Zhongling Lin, Ms. Guiying Yu, Ms. Tieling Ren at the department of Environmental Health in Public Health School of Sun Yat-sen University of Medical Sciences. I also thank my life time friend Mr. Wei Ren, Dr. Lei Yin, Dr. Xin Tong, and Dr. Tiger. During my Ph.D. study, their selfless friendships have been great support to me and will always be appreciated as the beauty of life. vii ABSTRACT mRNA degradation is a fundamental process that controls both the level and the fidelity of gene expression. Using a combination of bioinformatic, genomic, genetic, and molecular biology approaches, we have shown that Edc3p, a yeast mRNA decay factor, controls the stability of the intron-containing YRA1 pre-mRNA. We found that Edc3p-mediated degradation of YRA1 pre-mRNA: 1) is a component of a negative feedback loop involved in the autoregulation of YRA1, 2) takes place in the cytoplasm, 3) is independent of translation, 4) occurs through a deadenylation-independent decapping and 5΄ to 3΄ exonucleotic decay mechanism, and 5) is controlled by specific cis-acting elements and trans-regulatory factors. Cis-regulation of YRA1 pre-mRNA degradation is complicated and precise. Sequences in exon1 inhibit YRA1 pre-mRNA splicing and/or promote pre-mRNA export in a size-dependent but sequence-independent manner. Sequences in the intron dictate the substrate specificity for Edc3p-mediated decay. Five structurally different but functionally interdependent modules were identified in the YRA1 intron. Two modules, designated Edc3p-responsive elements (EREs), are required for triggering an Edc3p-response. Three other modules, designated translational repression elements (TREs), are required for repressing translation of YRA1 pre-mRNA. TREs enhance the efficiency of the response of the EREs to Edc3p by inhibiting viii translation-dependent nonsense-mediated mRNA decay (NMD). Trans-regulation of YRA1 pre-mRNA is governed by Yra1p, which inhibits YRA1 pre-mRNA splicing and commits the pre-mRNA to nuclear export, and the RNP export factors, Mex67p and Crm1p, which jointly promote YRA1 pre-mRNA export. Mex67p also appears to interact with sequences in the YRA1 intron to promote translational repression and to enhance the Edc3p response of YRA1 pre-mRNA.
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