The functions of the RNA polymerase II CTD in transcription and RNA processing Jing-Ping Hsin Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2013 © 2013 Jing-Ping Hsin All rights reserved Abstract The functions of the RNA polymerase II CTD in transcription and RNA processing Jing-Ping Hsin RNA polymerase II (RNAP II), transcribing messenger RNAs (mRNAs), small nuclear RNAs (snRNAs), and non-coding RNAs (ncRNAs), is composed of 12 subunits. Rpb1, the largest subunit with catalytic polymerase activity, possesses a unique c-terminal domain (CTD) that consists of tandem heptad repeats with the consensus sequence of Tyr-Ser-Pro-Thr-Ser-Pro-Ser (Y1S2P3T4S5P6S7). Somewhat reflecting the complexity of the organism, the number of repeats varies, from 26 in yeast to 52 in vertebrates. The CTD, intensively phosphorylated during transcription, serves a means to coordinate transcription and RNA processing- capping, splicing, and 3’ end formation. For example, Ser 5, phosphorylated in the start of transcription, promotes the recruitment of capping enzyme, and Ser 2 phosphorylation facilitates RNA 3’ end formation and transcription termination by acting as a landing pad for Pcf11. Detailed introduction is described in Chapter 1. Because of the importance of the CTD in these events, I created an Rpb1 conditional knock-out DT40 cell line (DT40-Rpb1) to further study the CTD with an initial focus on Thr 4, the function of which was unclear. Using DT40-Rpb1 system, we found that Thr 4 was phosphorylated in yeast, fly, chicken, and human cells, and cyclin-dependent kinase (CDK9) was likely the kinase to carry out this phosphorylation. We further provide evidence that Thr 4 functions in histone mRNA 3’ end formation (presented mostly in chapter 2 of this thesis). Chapter 3 mainly describes the studies regarding Ser 2, Ser 5, and Ser 7. Based on the DT40-Rpb1 cell line, I created stable cell lines expressing an Rpb1 carrying a CTD with Ser 2, Ser 5, or Ser 7 mutated to alanine, and investigated the phenotypes of these cells. We found that cells expressing an Rpb1 with S2A or S5A mutation were defective in transcription and RNA processing. Contrary to previous findings, we found Ser 7 was not involved in snRNA expression and 3’ end processing. In fact, no phenotypes associated with Ser 7 mutation were detected by our measurements. Extending previous Thr 4 studies, we showed in vitro and in vivo that Fcp1 dephosphorylated Thr 4. Finally, Chapter 4 describes what we have found the functions of CTD Tyr 1. Using the DT40-Rpb1 cells, I created stable cell lines expressing an Rpb1 with all Tyr residues mutated to phenylalanine (Phe). We found these cells were inviable, and the mutant Rpb1-Y1F was degraded to a CTD-less protein. Interestingly, the instability of Rpb1-Y1F was restored by reintroduction of one Tyr residue at the last heptad repeat. Further analysis provided evidence showing the involvement of Tyr phosphorylation in preventing Rpb1 from degradation by the 20S proteasome. Next, using ChIP assay, we showed Tyr phosphorylation was detected mostly at promoters, indicating a function of Tyr phosphorylation in transcription initiation. Indeed, transcription initiation defects were uncovered by assessing the recruitment of general transcription factors in cells with Y1F mutation. Extending this, we found an accumulation of upstream antisense RNAs in about one hundred reference genes by RNA-Seq analysis. TABLE OF CONTENTS Acknowledgements iv Dedication v Preface vi Chapter 1 The RNA polymerase II CTD coordinates transcription and RNA processing ………………………..….………… 1 Abstract ………………………………………………………………………………………………………………………………….…………… 2 Introduction ………………………………………………………………………………………………………………………….…………….. 2 CTD architecture and modification …………………………………………………………………………………….………………… 4 Transcription elongation, chromatin, and the CTD ……………………………………………………………………………… 13 RNA processing and the CTD ………………………………………………………………………………………………………………. 16 The CTD and transcription termination …………………………………………………………………………………………...…. 25 Perspectives …………………………………………………………………………………………………………………………………..…... 28 Acknowledgments ………………………………………………………………………………………………………………………………. 29 References ………………………………………………………………………………………………………………………………………….. 30 Figure legends …………………………………………………………………………………………………………………………………….. 48 Table …………………………………………………………………………………………………………………………………………………… 51 Figures …………………………………………………………………………………………………………………………………….……....... 53 Chapter 2 RNAP II CTD phosphorylated on threonine 4 is required for histone mRNA 3’ end processing ………... 58 i Abstract …………………………………………………………………………………………………………………………………………..… 59 Introduction …………………………………………………………………………………………………………………………………….… 60 Results ………………………………………………………………………………………………………………………………………………. 60 References and notes ………………………………………………………………………………………………………………………… 64 Acknowledgment ………………………………………………………………………………………………………………………………. 65 Figure legends …………………………………………………………………………………………………………………………………… 65 Figures …………………………………………………………………………………………………………………………………………….… 68 Supplemental materials ……………………………………………………………………………………………………………….……. 72 Chapter 3 Function and control of RNA polymerase II CTD in vertebrate transcription and RNA processing …… 91 Abstract …………………………………………………………………………………………………………………………………………..… 92 Introduction ……………………………………………………………………………………………………………………………………... 93 Results ………………………………………………………………………………………………………………………………………………. 96 Discussion …………………………………………………………………………………………………………………………………………. 102 Acknowledgment …………………………………………………………………………………………………………………..…….…… 107 Materials and methods …………………………………………………………………………………………………………………….. 107 References ……………………………………………………………………………………………………………………………………….. 110 Figure legends ………………………………………………………………………………………………………………………………….. 114 Table 1 ……………………………………………………………………………………………………………………………………………... 118 Supplemental figure legends …………………………………………………………………………………………..……………….. 118 Figures …………………………………………………………………………………………………………………………….……………….. 120 ii Supplemental figures ……………………………………………………………………………………………………………………….. 125 Chapter 4 Functional analysis of RNAP II CTD tyrosine 1 …………………………………………………………..…………………… 130 Abstract …………………………………………………………………………………………………………………..…………..………..… 131 Introduction …………………………………………………………………………………………………………..………………………... 132 Results …………………………………………………………………………………………………………………..…………………………. 135 Discussion …………………………………………………………………………………………………………………………………………. 143 Materials and methods …………………………………………………………………………………………………………………….. 148 Acknowledgment …………………………………………………………………………………………………………………..…….…… 151 References ……………………………………………………………………………………………………………………………………….. 151 Figure legends ………………………………………………………………………………………………………………………………….. 156 Supplemental figure legends …………………………………………………………………………………………..……………….. 159 Figures ……………………………………………………………………………………………………………………………………………… 161 Supplemental figures …………………………………………………………………………………………………………….…………. 167 iii Acknowledgements I would like first to thank my thesis research advisor, Jim Manley, for support over these years. From working in his lab, I have learned a great deal: how to write papers, defend ideas in the papers, and present data. I also like to thank my thesis committee members Larry Chasin and Carol Prives for support and many useful suggestions, and my other committee members, Liang Tong, Robert Fisher, and Stewart Shuman for taking the time to be on my thesis defense committee. I enjoy very much working in our very diverse lab with members of different backgrounds and ways of thinking. In the beginning of my project, I worked closely with Tsuyoshi Kashima, who taught me how to build the Rpb1 conditional knock-out cells. To him, I owe many thanks. I am thankful that I often get useful ideas through discussions with many lab members, especially Patricia Richard, Kehui Xiang, Ying Feng, Dafne Campigli Di Giammartino, Chuck David, and Emanuel Rosonina. I am grateful that Patricia, Chong Han Ng, Dafne, Tristan Coady, and Emanuel rehearsed many talks with me. Other lab members have been helpful in many ways- the assistance from Amit Sheth, Alex Boyne often voluntarily watching the levels of my running buffer, and etc. My family has been very supportive in this PhD journey. I appreciate vey much the encouragement they gave me when I was struggling with my research project. iv Dedication To my family v Preface This thesis is divided into four chapters. Chapter 1 is an introduction to the functions of the CTD of RNA polymerase II, and has been published as a review article in Genes and Development. Chapter 2 is a research article entitled “RNAP II CTD phosphorylated on threonine 4 is required for histone mRNA 3’ end processing,” published in Science. Chapter 3 is a manuscript with a title “Function and control of RNA polymerase II CTD phosphorylation in vertebrate transcription and RNA processing” that has been submitted for publication. Chapter 4 is the draft of a manuscript in preparation for publication. vi 1 Chapter 1 The RNA polymerase II CTD coordinates transcription and RNA processing Jing-Ping Hsin and James L. Manley Department of Biological Sciences Columbia University New York, NY 10027, USA 2 Abstract The carboxy terminal domain of the RNA polymerase II largest subunit (CTD) consists of multiple heptad repeats (consensus Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7), varying in number from 26 in yeast to 52 in vertebrates. The CTD functions to help couple transcription