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Molecular Analysis of Rna Polymerase Ii Elongation And MOLECULAR ANALYSIS OF RNA POLYMERASE II ELONGATION AND TERMINATION ON MAMMALIAN GENES by KRISTOPHER WADE BRANNAN B.A., University of Colorado Boulder, 2007 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Doctor of Philosophy Molecular Biology Program 2014 This thesis for the Doctor of Philosophy degree by Kristopher Wade Brannan has been approved for the Molecular Biology Program by Arthur Gutierrez-Hartmann, Chair David Bentley, Advisor Sandy Martin James DeGregori Thomas Evans Tom Blumenthal Date ___3/26/14________ ii Brannan, Kristopher Wade (Ph.D., Molecular Biology) Molecular Analysis of RNA Polymerase II Elongation and Termination on Mammalian Genes Thesis directed by Professor David Bentley ABSTRACT RNA polymerase II (pol II) pausing, elongation and termination are important mechanisms for controlling pol II distribution and transcriptional output during the transcription cycle on protein coding genes. This thesis focuses on mechanisms that influence pol II elongation through protein coding genes, and the state of elongating pol II during promoter proximal accumulation, elongation and termination. I address the following specific questions: 1) What factors are important for pol II termination? 2) What is the role of premature termination in limiting pol II elongation? 3) What is the role of CTD phosphorylation on pol II elongation and mRNA cleavage/polyadenylation? I report that decapping proteins and TTF2 interact with Xrn2 and that these factors localize by ChIP at 5’ ends of genes. Knockdown of decapping and termination factors by shRNA caused a widespread re-positioning of pol II at 5’ ends of genes away from start sites and toward distal positions both downstream and upstream. These results suggest that co-transcriptional decapping and premature termination by a torpedo mechanism is broadly employed to limit transcription of human genes. I also report that Fcp1 localizes at the 5’ end of human genes and limits CTD phosphorylation at both Ser2P and Ser5P positions. Fcp1 knockdown caused a widespread redistribution of pol II at gene 5’ ends away from transcription start sites (TSS) toward downstream positions, and localized increases in pol II Ser2P and Ser5P on highly expressed genes. Fcp1 iii knockdown also results in shifting in pA-site choice on ~1000 genes, primarily toward proximal positions. These results suggest that cotranscriptional dephosphorylation by Fcp1 is important for limiting both pol II elongation and usage of proximal alternative polyadenylation signals. Together these results have implications for new mechanisms regulating transcriptional control both at the elongation checkpoint and at the level of 3’ end formation. The form and content of this abstract are approved. I recommend its publication. Approved: David Bentley iv ACKNOWLEDGMENTS Thank you to the Bentley lab, to the Molecular Biology Program, to my family, and to Dr. Plank. v TABLE OF CONTENTS CHAPTER I. INTRODUCTION .................................................................................................. 1 1.1 Mechanisms of RNA pol II transcriptional pausing, elongation, and termination ............................................................................................................. 1 1.1A RNA pol II transcription cycle phases .................................................. 1 1.1B Termination models ............................................................................. 16 1.1C Xrn2 and associated proteins ............................................................... 22 1.1D Decapping ........................................................................................... 24 1.2 Regulation by co-transcriptional phosphatase activity .................................. 27 1.2A Dynamic phosphorylation of RNA pol II CTD Ser2 .......................... 28 1.2C Co-transcriptional phosphatase activity .............................................. 30 1.2D Alternative polyadenylation ................................................................ 31 1.3 Specific questions .......................................................................................... 33 II. MATERIALS AND METHODS .......................................................................... 34 2.1 Cell lines and growth conditions ................................................................... 34 2.2 Antibodies ..................................................................................................... 34 2.3 Immunopurification and mass spectrometry ................................................. 34 2.4 RNAi-mediated knock-downs ....................................................................... 35 2.5 RT-PCR ......................................................................................................... 35 2.6 ChIP, ChIP-Sequencing, mapping and analysis ............................................ 36 2.7 PA-Seq and analysis ...................................................................................... 37 2.8 Decapping reactions ...................................................................................... 38 vi III. mRNA DECAPPING FACTORS AND THE EXONUCLEASE XRN2 FUNCTION IN WIDESPREAD PREMATURE TERMINATION OF RNA POLYMERASE II TRANSCRIPTION ................................................................ 39 3.1 Summary ....................................................................................................... 39 3.2 Introduction ................................................................................................... 40 3.3 Results ........................................................................................................... 42 3.3A Xrn2 associates with TTF2 and mRNA decapping factors ................. 42 3.3B Termination and decapping factors localize at 5’ ends near paused pol II ................................................................................................................... 44 3.3C Depletion of Xrn2 and TTF2 re-distributes pol II away from the TSS toward promoter-distal positions ................................................................. 47 3.3D Decapping factor knockdown redistributes pol II away from promoter- proximal positions ........................................................................................ 59 3.3E Comparing the roles of termination, decapping, and pausing factors in pol II localization ......................................................................................... 63 3.3F Knockdown of termination and decapping factors redistributes pol II within ‘‘pausing-regulated’’ genes .............................................................. 65 3.4 Discussion ..................................................................................................... 68 3.4A A Nuclear function for decapping factors in promoting premature termination of transcription .......................................................................... 68 3.4B Premature termination and the control of eukaryotic gene expression .................................................................................................... 70 IV. COTRANSCRIPTIONAL DEPHOSPHORYLATION OF RNA POL II INFLUENCES PROMOTER PROXIMAL PAUSING AND PA-SITE CHOICE ................................................................................................................ 74 4.1 Abstract ......................................................................................................... 74 4.2 Introduction ................................................................................................... 75 4.3 Results ........................................................................................................... 78 4.3A Fcp1 localizes at 5’ ends near paused pol II ....................................... 78 vii 4.3B Depletion of Fcp1 reduces promoter proximal pol II pausing ............ 78 4.3C Fcp1 knockdown increases relative CTD Ser2P and Ser5P within gene bodies ........................................................................................................... 82 4.3D Fcp1 knockdown results in an upstream shift in pA-site choice ......... 88 4.3E Fcp1 knockdown does not alter recruitment of Cstf77 ....................... 93 4.4 Discussion ..................................................................................................... 97 V. CONCLUSIONS/DISCUSSION ........................................................................ 101 REFERENCES ............................................................................................................... 108 APPENDIX A. SUPPLEMENTAL TABLES ................................................................................... 129 viii LIST OF TABLES TABLE 1. Human pLKO.1 lentiviral shRNA ............................................................................. 129 2. Proteins identified by mass spectrometry .................................................................. 130 3. PSY analysis of pA-site shifts ................................................................................... 132 ix LIST OF FIGURES FIGURE 1-1 The pol II transcription cycle proceeding through three phases..................................2 1-2 Stepwise assembly of the pre-initiation complex (PIC)...............................................4 1-3 RNA pol II density profile across a typical metazoan protein-coding gene.................7 1-4 Promoter proximal pausing
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