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Ribosome Profiling of Late Adenovirus Infected Cells Reveals Ribosome Stacking on the 5’ Untranslated Region of Late Messenger Rna

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Ribosome Profiling of Late Adenovirus Infected Cells Reveals Ribosome Stacking on the 5’ Untranslated Region of Late Messenger Rna RIBOSOME PROFILING OF LATE ADENOVIRUS INFECTED CELLS REVEALS RIBOSOME STACKING ON THE 5’ UNTRANSLATED REGION OF LATE MESSENGER RNA BY JASON M. GAGLIANO A Dissertation Submitted to the Graduate Faculty of WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Biology August 2019 Winston-Salem, North Carolina Approved By: James F. Curran, Ph.D., Advisor David A. Ornelles, Ph.D., Chair Keith Bonin, PhD. Erik C. Johnson, Ph.D. Ke Zhang, Ph.D. ACKNOWLEDGEMENTS I would like to thank Dr. Jim Curran, Dr. David Ornelles, and Dr. Keith Bonin for the endless support I received from them on this challenging project. I am grateful for all the time and effort they spent in helping me work through problems in bench chemistry, biological assays and bioinformatics. I would also like to thank my entire committee for their insights and helpful suggestions towards making this project a success. Furthermore, I thank the entire biology department of Wake Forest University for accepting me and allowing me to embark on a part-time Ph.D. project. In addition, I would like to thank Dr. Jennifer Weller (UNC Charlotte) for her training in high-throughput sequencing and bioinformatics. Of course, I need to thank my wife Zeynia and my three children, Kayla, Antonio and Alyanna for their patience as I worked endless hours on my project. Finally, I would like to thank all those who indirectly supported me through this project and whose contributions were important tomy success: Dr. Jed Macosko (WFU physics), Dr. Martin Guthold (WFU physics), Dr. Roger Cubicciotti (NanoMedica LLC), Kara Libby (NanoMedica LLC), Dr. George Holzwarth (WFU physics), Dr. Nicholas Ingolia (Berkeley), Dr. Eric Davies (NC State), Dr. Hugh Bender (UC Irvine), and Dr. David Matthews (University of Bristol). II TABLE OF CONTENTS ACKNOWLEDGEMENTS ............................................................................................ II TABLE OF CONTENTS ............................................................................................... III LIST OF TABLES ........................................................................................................ VII LIST OF FIGURES ..................................................................................................... VIII LIST OF ABBREVIATIONS ........................................................................................ XI ABSTRACT .................................................................................................................. XIV INTRODUCTION ............................................................................................................ 1 The Current Understanding of Adenovirus Infection ..................................................... 1 The Early Phase of Adenovirus Infection ....................................................................... 3 The Delayed Early and Late Phase of Adenovirus Infection .......................................... 4 Transcription and Post-Transcription Processing of HAdVC mRNA ............................ 5 Cap-Dependent Translation of Host, Viral Early and IX mRNA ................................... 9 The Preferential Translation of Tripartite and IVa2 mRNA via Ribosome Shunting .. 13 The L4 100K Protein .................................................................................................... 14 18S rRNA Complementarity ........................................................................................ 17 Ribosome Shunting ....................................................................................................... 19 The Translation of Circular mRNA .............................................................................. 22 Polyribosome and Ribosome Profiling ......................................................................... 23 Hypothesis for the Translation of Late Shunting-mRNA ............................................. 26 MATERIALS AND METHODS ................................................................................... 28 Cell Culture ............................................................................................................... 28 Virus .......................................................................................................................... 28 Viral Infection ........................................................................................................... 29 III Sucrose Gradients ..................................................................................................... 29 Cell Lysis .................................................................................................................. 30 Polysome Profiling .................................................................................................... 30 Ribosomal RNA Purification from Sucrose Fractions .............................................. 31 Control Ribosome Protected Fragments ....................................................................... 31 RNA Separation by Denaturing Polyacrylamide Gel Electrophoresis ......................... 32 RNA Quantification by Gel Densitometry ................................................................... 32 RNA Separation and Quantification by Microchip Electrophoresis ............................ 33 Control RNA Precipitation from Polysome Fractions .............................................. 34 Control RNA Enrichment from Polysome Fractions ................................................ 36 Control RNA Polyacrylamide Gel Purification ........................................................ 38 Ribosome Profiling ................................................................................................... 40 Polynucleotide Kinase Modification of Ribosome Protected Fragments ................. 42 Reverse Transcription of Ribosome-protected Fragment ......................................... 42 Template Preparation for Sequencing ....................................................................... 45 The Measured Distribution of Templated vs Non-Templated Ion Sphere Particles . 45 Enrichment of Template-Positive ISPs ..................................................................... 46 High Throughput Sequencing of the cDNA library Using the IonTorrent Personal Genome Machine ...................................................................................................... 47 Integrity Check of the FASTQ File After Downloading .......................................... 47 Read-Length Determination of Sequencing Reads ................................................... 48 Quality Scoring of Sequencing Output ..................................................................... 48 Quality Trimming, Filtering and Mapping ............................................................... 49 RNA-Seq Mapping with CLC .................................................................................. 50 The Detection of Periodicity from Mapped Reads ................................................... 50 IV RESULTS ........................................................................................................................ 51 Separation and Detection of Polyribosomes in Translation .......................................... 51 Verification of RNA and Ribosomal RNA from the Polysome Profile ....................... 53 Separation and Detection of Single 80S Ribosomes in Translation ............................. 54 Polyribosome and Ribosome Profile of Adenovirus Infected HeLa Cells ................... 56 High Throughput Sequencing of Ribosome-protected Fragments from Adenovirus Infected Cells ................................................................................................................ 57 Trimming or Filtering ................................................................................................... 58 Mapping Sequences to the HAdVC Reference Genome AY601635 ........................... 61 Mapping of RPFs to HAdVC, Human, rRNA, and HPV-18 References ..................... 61 Translational Phenomena .............................................................................................. 64 Spliced Messages .......................................................................................................... 64 Reading Frames ............................................................................................................ 65 Periodicity ..................................................................................................................... 65 Mapping to the Adenovirus Genome to Study Late Translation .................................. 66 The Analysis of 80S Ribosomes in the 5’ UTR of Shunting-mRNA ........................... 69 Ribosome Protection of 18S rRNA Complementary regions in the 5’ UTR of TPL and IVa2-mRNA Exhibit Hierarchical Density of 80S Ribosomes ............................. 70 Mapping to the Individual Adenovirus mRNA to Study Late Translation ................... 71 Comparison of Ribosome Profile vs RNA-Seq of Adenovirus Infected Cells ............. 72 DISCUSSION .................................................................................................................. 74 Models of Canonical Translation and Shunting ........................................................... 74 80S Ribosomes Appear to Stack in the 5’ UTR of Shunting-mRNA ........................... 76 Ribosomes in the 5’ UTR of Shunting-mRNA do not Appear
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