Identification of Cellular RNA Binding Sites of DEAD-Box Helicases
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IDENTIFICATION OF CELLULAR RNA BINDING SITES OF DEAD-BOX HELICASES By FRANK TEDESCHI Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation Advisor: Eckhard Jankowsky Department of Biochemistry CASE WESTERN RESERVE UNIVERSITY August, 2018 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Frank Tedeschi candidate for the degree of Doctorate of Philosophy*. Committee Chair Dr. Donny Licatalosi Committee Members Dr. Eckhard Jankowsky Dr. Jeff Coller Dr. William Merrick Dr. Derek Taylor Date of Defense 6-13-18 *We also certify that written approval has been obtained for any proprietary material contained therein. 2 Table of Contents Acknowledgements ............................................................................................ 11 List of Abbreviations ........................................................................................... 13 Abstract .............................................................................................................. 16 Chapter One RNA Helicases General Structure, Biochemical., and Biological Function 18 1.1 Introduction ................................................................................................ 18 1.2 Biological Functions of DEAD-box helicases ......................................... 19 1.2.1 Transcription .............................................................................................. 20 1.2.2 Nuclear Export ........................................................................................... 20 1.2.3 Translation ................................................................................................. 21 1.2.4 Ribosome Biogenesis ................................................................................ 22 1.3 RNA Helicases Families and Structure .................................................... 24 1.4 Enzymatic Activities of DEAD-box Helicases ......................................... 26 1.4.1 RNA unwinding by local strand separation ................................................ 27 1.4.2 ATPase activity .......................................................................................... 29 1.4.3 RNA clamping, annealing, and remodeling................................................ 30 1.5 Introduction to Crosslinking Immunoprecipitation (CLIP) ..................... 32 1.6 Comparison of HITS-CLIP, PAR-CLIP, iCLIP, and dCLIP ....................... 36 1.6.1 HITS-CLIP ................................................................................................. 36 1.6.2 PAR-CLIP .................................................................................................. 37 1.6.3 iCLIP .......................................................................................................... 39 1.6.4 eCLIP......................................................................................................... 41 1.6.5 Denaturting CLIPs ..................................................................................... 43 1.6.6 RNA Helicases and CLIP ........................................................................... 45 Chapter Two The DEAD-box helicases Dbp2p is linked to non-coding RNAs, the helicase Sen1p, and R-loops ........................................................................... 49 2.1 Introduction to the DEAD-box helicase Dbp2p ....................................... 49 2.2 Results ........................................................................................................ 51 2.2.1 iCLIP of Dbp2p-HTBH ............................................................................... 51 3 2.2.2 Dbp2p crosslinks to non-coding RNAs ...................................................... 57 2.2.3 Dbp2p binds to 3’ proximal regions in snoRNAs ........................................ 58 2.2.4 Dbp2p binding to snoRNAs reveals link to R-loops and the RNA helicase Sen1p ................................................................................................................. 68 2.2.5 Dbp2p binding to tRNA indicates further links to R-loops .......................... 73 2.2.6 Dbp2p binding to rRNA, mRNA, snRNA, and TY elements indicates additional links to R-loops ................................................................................... 79 2.3 Conclusions ............................................................................................... 88 Chapter Three The helicase Ded1p controls use of near-cognate translation initiation codons in 5’ UTRs ............................................................................................ 90 3.1 Introduction to the DEAD-box RNA helicase Ded1p ............................... 90 3.2 Defects in Ded1p activate alternative translation initiation sites ........... 94 3.3 mRNA structure unwinding by Ded1p and aTIS activation ..................... 99 3.4 Individual nucleotide resolution crosslinking immunoprecipitation (iCLIP) of Ded1p .............................................................................................. 102 3.4.1 Tandem Affinity Purification of Ded1p-HTBH ........................................... 103 3.4.2 Ded1p-HTBH cDNA library generation .................................................... 105 3.4.3 Ded1p-HTBH crosslinking to the ribosome and to mRNA ....................... 109 3.5 Alterations in PSA1 and ATP5 mRNA show direct link between Ded1p unwinding and aTIS activation .......................................................... 121 3.6 A model for Ded1p function in translation initiation ............................. 123 3.7 Ded1p is repressed during meiosis which exhibits extensive aTIS activation ......................................................................................................... 125 3.8 Conclusions ............................................................................................. 128 Chapter Four Future Directions ............................................................................................ 129 4.1 Introduction ............................................................................................... 129 4.2 Ded1p in translation initiation ................................................................ 129 4.3 Dbp2p, Sen1p, and R-loop resolution .................................................... 130 4.4 Ribosomal modifications dependent on Dbp2p ................................... 131 4.5 snoRNA changes under the cellular stress of glucose depletion ....... 132 4 4.6 Characterization of cold sensitive mutant Ded1p-G368D and the effects of nutrient source............................................................................... 133 4.6.1 Results..................................................................................................... 134 4.6.2 Conclusions ............................................................................................. 136 Chapter Five Materials and Methods ................................................................................... 137 5.1 Yeast strains, plasmids, and oligonucleotides ...................................... 137 5.2 Polysome analysis ................................................................................... 137 5.3 Westeron blot analysis............................................................................. 138 5.4 Ded1p-HTBH iCLIP ................................................................................... 138 5.5 Bioinformatic analyses ........................................................................... 141 5.6 Unwinding Reactions .............................................................................. 142 5.7 Generation of Dbp2-HTBH strain ............................................................ 142 5.8 Dbp2p iCLIP .............................................................................................. 143 5.9 Bioinformatic Analysis ............................................................................. 148 5.10 RNA-seq .................................................................................................. 150 5.11 Co-immunoprecipitation ....................................................................... 151 Appendix A: R code ....................................................................................... 156 A1: Table creation that reflects all chromosomes and nucleotides for yeast ... 156 A2: Binding density surrounding the 3’ end of transcripts ................................. 161 A3: Generation of Metagene profiles for snoRNAs ........................................... 196 A4: Generatin of Metagene profiles from transcription start site to polyadenylation site for mRNAs ........................................................................ 223 A5: Extracting the binding density of Sen1p or R-loop surround Dbp2p binding peaks ................................................................................................... 256 A6: EdgeR differential expression of RNA-seq datasets ................................. 290 A7: RNA-seq fold change expression violin plots ............................................ 292 Bibliography .................................................................................................... 297 5 List of Figures Chapter One Figure 1. SF1 and SF2 helicase families ......................................................... 19 Figure 2. Architecture of the DEAD-box helicases .......................................... 24 Figure