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Evidence for Coupling Transcription and Splicing in Vivo in Saccharomyces Cerevisiae

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Evidence for Coupling Transcription and Splicing in Vivo in Saccharomyces Cerevisiae EVIDENCE FOR COUPLING TRANSCRIPTION AND SPLICING IN VIVO IN SACCHAROMYCES CEREVISIAE DISSERTATION Presented in Partial Fulfillment of the Requirement for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Luh Tung, M.S. * * * * * The Ohio State University 2007 Dissertation Committee: Dr. Tien-Hsien Chang, Advisor Dr. Venkat Gopalan Dr. Paul K. Herman Dr. Amanda Simcox ABSTRACT This dissertation describes the study of two DExD/H-box proteins in the budding yeast Saccharomyces cerevisiae: The first part (Chapter 1 to 4) outlines the discovery that specific alterations can bypass the requirement of Sub2p, an essential DExD/H-box protein that functions in precursor messenger RNA (pre-mRNA) splicing and in coupling transcription to mRNA export. However, its precise mode of action remains to be defined. Here, I show that the otherwise essential Sub2p can be made dispensable by specific alterations of the intron branch-site-binding protein (BBP) within its conserved branch-site recognition domain. This result suggests that Sub2p acts as a Ribonucleoprotein ATPase (RNPase) to remodel the splicing complex by removing BBP from the branch site, thereby allowing subsequent binding of the U2 snRNP. Unexpectedly, specific alterations of several transcription factors, as well as perturbing transcription elongation by 6-arauracil (6-AU), can also eliminate the requirement of Sub2p. Chromatin-immunoprecipitation (ChIP) experiments revealed that these perturbations significantly reduce the co-transcriptional recruitment of BBP, thus offering a satisfactory explanation as to how Sub2p is bypassed. Most ii significantly, these results provide compelling evidence that transcription and splicing in yeast are coupled and that this strategy may be conserved. The second part (Chapter 5) documents the genetic characterization of Ded1p, an evolutionarily conserved DExH/D-box protein in the budding yeast. Ded1p is indispensable for translation, but it is also functionally linked to pre-mRNA splicing and virus propagation. In this Chapter, I report a novel aspect of Ded1p’s functions. I first showed that combinations of mutant ded1 alleles with a deletion allele of TIF4631, which encodes one of the two eIF4G translation initiation factors, resulted in a synthetic-lethal growth phenotype. Unexpectedly, an open-ended search led to the identification of RTG3, which encodes a component involved in the retrograde (RTG) signaling pathway employed in yeast for responding to mitochondrial dysfunction. Further analyses revealed that this synthetic-lethal phenotype is related to RTG1, RTG2, and RTG3, which are required for turning on the glyoxylate cycle. However, deletion of other genes involved in the glyoxylate cycle did not result in the same lethal phenotype. Consistent with the rapamycin-resistant phenotype exhibited by rtg mutants, ded1 mutants are also rapamycin resistant, thereby suggesting a relationship of Ded1p to the TOR (target of rapamycin) signaling pathway. Since the TOR pathway was reported to control the protein stability of eIF4G, Ded1p may also be involved in regulating eIF4G level via the TOR signaling pathway. iii Dedicated to my parents iv ACKNOWLEDGMENTS I thank my advisor Dr. Tien-Hsien Chang for providing me with an excellent training environment with intellectual support and guidance during my graduate school career. I also thank Dr. Venkat Gopalan, Dr. Paul K. Herman, Dr. Amanda Simcox, and Dr. Lee F. Johnson for their helpful discussions, suggestions and encouragements as my committee members. I wish to thank all members of the Chang laboratory especially Rosemary Hage and Dr. Jean-Leon Chong for their friendship, support and assistance. I am grateful to Hsin-yue, Liz Oakley, Marianne, and Dr. Michael Chan for their encouragement and support during these years. Thanks also go to Dr. Christine Guthrie, Dr. Michael Hampsey, Dr. Michael Rosbash, Dr. Bertrend Séraphin, Dr. Ed Hurt and Dr. Grant A. Hartzog for providing reagents. Finally, I am deeply indebted to my dear parents, brother and Li-chi Chang for their support, encouragement and understanding during my graduate school years. v VITA Oct 1975.......................................... Born – Taipei, Taiwan July 1997.......................................... B.S. in Department of Zoology, National Taiwan University, Taiwan July 1999.......................................... M.S. in Institute of Molecular Medicine, National Taiwan University, Taiwan 1999-present.................................... Graduate Research and Teaching Associate, Graduate Program in Molecular, Cellular and Development Biology (MCDB), The Ohio State University, Columbus, OH, United States PUBLICATIONS Pryor, A., Tung, L., Yang, Z., Kapadia, F., Chang, T.-H. and Johnson, L.F. (2004). Growth-regulated expression and G0-specific turnover of the mRNA that encodes URH49, a mammalian DExH/D box protein that is highly related to the mRNA export protein UAP56. Nucleic Acids Research 32, 1857-1865. vi Chong, J.-L., Chuang, R.-Y., Tung, L. and Chang, T.-H. (2004). Ded1p, a conserved DExD/H-box translation factor, can promotoe yeast L-A virus negative-strand RNA synthesis in vitro. Nucleic Acids Research 32, 2031-2038. Huang, C.F., Liu, Y.W., Tung, L., Lin, C.H. and Lee FJ. (2003) Role for Arf3p in development of polarity, but not endocytosis, in Saccharomyces cerevisiae. Mol. Biol. Cell 14, 3834-3847. Huang, C.F., Chen, C.C., Tung, L., Buu, L.M., and Lee, F.J. (2002) The yeast ADP-ribosylation factor GAP, Gcs1p, is involved in maintenance of mitochondrial morphology. J. Cell Sci. 115(Pt 2), 275-282. FIELDS OF STUDY Major Field: Molecular, Cellular, and Developmental Biology vii TABLE OF CONTENTS Page Abstract ................................................................................................................ ii Dedication ............................................................................................................ iv Acknowledgments................................................................................................. v Vita ...................................................................................................................... vi List of Tables ....................................................................................................... xiii List of Figures ..................................................................................................... xv Chapters: 1. INTRODUCTION .............................................................................................. 1 1.1 Pre-mRNA Splicing ...................................................................................... 2 1.2 The Spliceosome ......................................................................................... 4 1.3 The Dynamic Nature of the Spliceosome .................................................... 5 1.3.1 Assembly of the Spliceosome ........................................................... 5 1.3.2 Recognition of the Intron-Branch Site by BBP and Mud2p ................ 7 1.3.2.1 Branch Site Binding Protein (BBP) ........................................ 7 1.3.2.2 U2 Auxiliary Factor (U2AF) and Mud2p ............................... 10 1.3.3 DExD/H-box Proteins Function as RNPase in Splicing ................... 13 1.3.4 UAP56 and Sub2p .......................................................................... 14 1.3.4.1 Sub2p Functions in pre-mRNA Splicing .............................. 15 1.3.4.2 Sub2p Functions in mRNA Export ....................................... 16 1.4 Gene Expression Pathways Are Tightly Coupled ...................................... 16 1.4.1 The RNA polymerase II (Pol II) C-terminal domain (CTD) Serves as a Recruitment Platform ............................................................. 18 viii 1.4.2 Transcription and Splicing Are Coupled........................................... 19 1.4.3 Chromatin Immunoprecipitation (ChIP) ........................................... 21 1.5 Goal of This Work...................................................................................... 22 2. MATERIALS AND METHODS ........................................................................ 27 2.1 Yeast Strains ............................................................................................. 27 2.2 Plasmids.................................................................................................... 27 2.3 Oligos ........................................................................................................ 27 2.4 Analysis of msl5 Alleles Capable of Bypassing sub2 .............................. 27 2.5 Genetic Screening of the sub2 Bypass Mutants ..................................... 29 2.6 Southern blot analysis of SUB2 alleles of the sub2 Bypass Mutants ...... 29 2.7 Genetic characterization of the sub2 Bypass Mutants ............................ 30 2.8 Identification of the sub2 Bypass Mutants ............................................... 31 2.9 Perturbations of the Transcription Machinery ............................................ 31 2.10 Examination of the capability of prp40-1 to bypass sub2 ...................... 33 2.11 Examination of the Requirement of SUB2 in Medium Containing Different Carbon Sources ....................................................................... 33 2.12 Examination of the Genetic Interaction between SUA7 and MUD2 ......... 34 2.13 Examination of the Genetic Interaction Between MSL5 and MUD2 ........ 34 2.14 Examination of the Genetic Interaction
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