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Termination of RNA Polymerase II Transcription by the 5’-3’ Exonuclease Xrn2

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Termination of RNA Polymerase II Transcription by the 5’-3’ Exonuclease Xrn2 TERMINATION OF RNA POLYMERASE II TRANSCRIPTION BY THE 5’-3’ EXONUCLEASE XRN2 by MICHAEL ANDRES CORTAZAR OSORIO B.S., Universidad del Valle – Colombia, 2011 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 2018 This thesis for the Doctor of Philosophy degree by Michael Andrés Cortázar Osorio has been approved for the Molecular Biology Program by Mair Churchill, Chair Richard Davis Jay Hesselberth Thomas Blumenthal James Goodrich David Bentley, Advisor Date: Aug 17, 2018 ii Cortázar Osorio, Michael Andrés (Ph.D., Molecular Biology) Termination of RNA polymerase II transcription by the 5’-3’ exonuclease Xrn2 Thesis directed by Professor David L. Bentley ABSTRACT Termination of transcription occurs when RNA polymerase (pol) II dissociates from the DNA template and releases a newly-made mRNA molecule. Interestingly, an active debate fueled by conflicting reports over the last three decades is still open on which of the two main models of termination of RNA polymerase II transcription does in fact operate at 3’ ends of genes. The torpedo model indicates that the 5’-3’ exonuclease Xrn2 targets the nascent transcript for degradation after cleavage at the polyA site and chases pol II for termination. In contrast, the allosteric model asserts that transcription through the polyA signal induces a conformational change of the elongation complex and converts it into a termination-competent complex. In this thesis, I propose a unified allosteric-torpedo mechanism. Consistent with a polyA site-dependent conformational change of the elongation complex, I found that pol II transitions at the polyA site into a mode of slow transcription elongation that is accompanied by loss of Spt5 phosphorylation in the elongation complex. Inhibition of polyA signal recognition by expression of the flu virus NS1A protein or CRISPR mediated mutation of a consensus polyA signal further revealed that binding of 3’-end processing factors to the polyA signal on the RNA transcript is required for both deceleration of pol II and loss of Spt5 phosphorylation downstream of polyA sites. Consistent with the torpedo model, I report that the 5’ PO4 RNA end generated at the polyA cleavage site accumulates to high levels when the exonuclease activity of Xrn2 is inhibited. The primary product of polyA site cleavage is therefore directly degraded by Xrn2. Furthermore, inhibition of Xrn2 resulted in accumulation of iii polymerases downstream of polyA sites that fail to dissociate from the DNA template after a switch to slow elongation. I propose an allosteric-torpedo model in which polyA site-dependent transcriptional deceleration by 3’-end processing factors permits the Xrn2 torpedo to catch pol II and dismantle the elongation complex. Additionally, I found that premature termination of transcription is a common phenomenon on human promoters and that Xrn2 participates in premature termination of a fraction of polymerases that can elongate into gene bodies without licensing by pTEF-b. Consistent with a role of Xrn2 in premature termination, I found that Xrn2 is recruited to gene promoters and migrates to 3’ ends of genes associated with the elongation complex. Finally, I investigated R-Loop structures genome-wide and provide evidence that mammalian cells possess a protective mechanism against accumulation of R-Loops on pol II transcribed genes to prevent their potential threat to genomic instability. The form and content of this abstract are approved. I recommend its publication. Approved: David L. Bentley iv GRAPHICAL ABSTRACT TERMINATION OF RNA POLYMERASE II TRANSCRIPTION BY THE 5’-3’ EXONUCLEASE XRN2 v TABLE OF CONTENTS CHAPTER I. INTRODUCTION ................................................................................................................ 1 1.1 Transcription by the Different Eukaryotic RNA Polymerases ..................................... 1 1.2 Transcription by RNA Polymerase II .......................................................................... 6 1.3 Promoter-Proximal Pausing of Pol II .......................................................................... 6 1.4 The Elongation Factor Spt5 ...................................................................................... 13 1.5 mRNA 3’-end Processing ......................................................................................... 15 1.6 The 5’-3’ Exonuclease Xrn2 ..................................................................................... 18 1.7 Termination of Pol II Transcription .......................................................................... 21 1.8 The Transcriptional Response to Heat Shock ............................................................ 29 1.9 R-Loop Structures During Transcription by RNA pol II ............................................ 31 II. MATERIALS AND METHODS ....................................................................................... 37 2.1 Chromatin Immunoprecipitation Coupled to Sequencing (ChIP-seq) ........................ 37 2.1A Chromatin Immunoprecipitation (ChIP) ......................................................... 37 2.1B ChIP-seq and Mapping ................................................................................... 38 2.2 Antibodies ................................................................................................................ 39 2.3 Bromouridine Sequencing (Bruseq) .......................................................................... 39 2.3A Isolation of Nascent Transcripts ..................................................................... 39 2.3B Bruseq and Mapping ...................................................................................... 40 vi 2.4 5’-PO4 Bromouridine Sequencing (5’-Bruseq) .......................................................... 41 2.5 DNA-RNA Immunoprecipitation Coupled to Sequencing (DRIP-seq) ...................... 44 2.6 Engineering of pcDNA5-NS1A Inducible Flp-in TREX Cells .................................. 44 2.7 CRISPR-Cas9 Genome Editing of the PSMG1 polyA Signal in Hap1 Cells .............. 45 2.8 Doxycycline-Inducible shRNA Expression of Xrn2 and TTF2 in HEK293 Cells ...... 48 2.9 Cell Lines Growth Conditions .................................................................................. 51 2.10 Real-Time quantitative (q) PCR .............................................................................. 51 III. INTEGRATION OF XRN2 INTO THE TRANSCRIPTION CYCLE OF RNA POLYMERASE II ........................................................................................................... 54 3.1 Introduction .............................................................................................................. 54 3.2 Results ...................................................................................................................... 58 3.2A Recruitment of Xrn2 Around Promoters does not Require Synthesis of a pre- mRNA Transcript .......................................................................................... 58 3.2B Xrn2 Associates with Promoter-Proximally Paused Elongation Complexes .... 60 3.2C Xrn2 Accompanies the Elongation Complex from Promoters to 3’ Ends of Genes ............................................................................................................ 63 3.2D Xrn2 Degrades Nascent Transcripts Downstream and Upstream of polyA Sites ...................................................................................................................... 72 3.2E 5’-Bruseq Detects 5’-Monophosphorylated Ends of Nascent Transcripts ........ 77 3.2F Xrn2 Recognizes the 5’ PO4 End of Nascent Transcripts at polyA Sites for Degradation .................................................................................................... 83 3.2G Xrn2 Degrades the Nascent Transcript Downstream of the pre-rRNA 3’ End . 87 3.2H Xrn2 Targets the 5’ End of snRNAs ............................................................... 90 vii 3.3 Discussion ................................................................................................................ 93 IV. THE ALLOSTERIC-TORPEDO MODEL OF RNA POL II TRANSCRIPTION TERMINATION .............................................................................................................. 99 4.1 Introduction .............................................................................................................. 99 4.2 Results .................................................................................................................... 105 4.2A Xrn2 is Required for Release of pol II from the DNA Template Downstream of polyA Sites .................................................................................................. 105 4.2B Pol II Decelerates Transcription Elongation Downstream of polyA Sites ...... 110 4.2C Slowdown of pol II Downstream of polyA Sites Requires Recognition of the polyA Signal................................................................................................ 117 4.2D Spt5 Transitions Into a Hypo-Phosphorylated State in the Elongation Complex Downstream of polyA Sites ......................................................................... 125 4.2E Recognition of the polyA Signal is Required for Loss of Spt5 Phosphorylation Downstream of polyA Sites ......................................................................... 133 4.3 Discussion .............................................................................................................
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