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The Panicum Mosaic Virus-Like 3' Cap-Independent Translation Element

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The Panicum Mosaic Virus-Like 3' Cap-Independent Translation Element Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2013 The aP nicum mosaic virus-like 3' cap-independent translation element: A translation enhancer that functions in mammalian systems Mariko Sada Peterson Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Biochemistry Commons, Molecular Biology Commons, and the Virology Commons Recommended Citation Peterson, Mariko Sada, "The aP nicum mosaic virus-like 3' cap-independent translation element: A translation enhancer that functions in mammalian systems" (2013). Graduate Theses and Dissertations. 13061. https://lib.dr.iastate.edu/etd/13061 This Thesis is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. The Panicum mosaic virus-like 3’ cap-independent translation element: A translation enhancer that functions in mammalian systems by Mariko Sada Peterson A thesis submitted to the graduate faculty in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Major: Biochemistry Program of Study Committee: W. Allen Miller, Major Professor Susan Carpenter Mark Hargrove Iowa State University Ames, Iowa 2013 Copyright © Mariko Sada Peterson, 2013. All rights reserved. ii TABLE OF CONTENTS ACRONYMS USED IN THIS THESIS: ........................................................................................ iv ABSTRACT ..................................................................................................................................... v CHAPTER 1. GENERAL INTRODUCTION: A REVIEW OF THE LITERATURE ................. 1 Historical Perspective .................................................................................................................. 1 Canonical translation initiation in plants and mammals ............................................................. 2 Virus strategies of translation initiation: IRESes and CITEs ...................................................... 8 The Panicum mosaic virus-like translation element (PTE), an eIF4E-binding CITE ............... 14 eIF4E in plants and mammals ................................................................................................... 16 The Tombusviridae and the Umbraviruses................................................................................ 20 Project Aims .............................................................................................................................. 25 CHAPTER 2: MATERIALS AND METHODS .......................................................................... 26 eIF4E Sequence and Structure Alignment ................................................................................ 26 RNA Preparation ....................................................................................................................... 26 T7-BHK cell culture and DNA transfection ............................................................................. 28 HeLa cell culture and RNA transfection ................................................................................... 28 In-vitro translation assays.......................................................................................................... 29 CHAPTER 3: RESULTS AND DISCUSSION ............................................................................ 32 eIF4E sequence and structural alignments ................................................................................ 32 Aim 1: System Optimization ..................................................................................................... 36 Aim 2: Performance of PTE-containing luciferase reporters in lysates and cells ..................... 45 Aim 3: Performance of the PTE as an inhibitor ........................................................................ 52 CHAPTER 4: CONCLUSIONS ................................................................................................... 55 Relevance .................................................................................................................................. 55 Proposed model ......................................................................................................................... 56 Future directions ........................................................................................................................ 56 APPENDIX 1: 3’ TRUNCATION OF PTE CONSTRUCTS ...................................................... 58 APPENDIX 2: PREPARATION OF HELA CELL EXTRACT .................................................. 60 iii APPENDIX 3: APPENDIX 3: EFFECT OF POTASSIUM IONS ON BTE TRANS INHIBITION................................................................................................................................. 62 REFERENCES CITED ................................................................................................................. 64 ACKNOWLEDGEMENTS .......................................................................................................... 79 iv ACRONYMS USED IN THIS THESIS: BHK: Baby hamster kidney (cell line) BTE: Barley yellow dwarf virus-like cap-independent translation element CarMV: Carnation mottle virus CITE: Cap-independent translation enhancer CMMV: Cocksfoot mild mosaic virus EMCV: Endomyocarditis virus HCRSV: Hibiscus ring spot virus IRES: Internal ribosome entry site JINRV: Japanese iris necrotic ringspot virus MNSV: Melon necrotic spot virus PEMV-2: Pea enation mosaic virus RNA-2 PFBV: Pelagornium flower break virus PIC: Pre-initiation complex PMV: Panicum mosaic virus PTE: Panicum mosaic virus-like cap-independent translation element RRL: Rabbit reticulocyte lysate SCV: Saguaro cactus virus TPAV: Thin paspalum asymptomatic virus UTR: Untranslated region VPg: Viral protein, genome-linked WGE: Wheat germ extract v ABSTRACT In canonical eukaryotic translation initiation, the recognition of mRNA by the translation machinery is facilitated through the binding of the 5’ m7G cap structure to eukaryotic initiation factor 4E (eIF4E). This recognition is necessary for efficient translation of the message to occur. In contrast, some viruses lack the 5’ cap structure but have alternative mechanisms to initiate translation and express the viral proteins necessary for replication. The positive-strand RNA plant viruses of the panico-, carmo-, umbra-, (1-3) and aureusviruses (4) contain an RNA structure in their 3’ UTRs that binds eIF4E (2) and through communication with the 5’ UTR, are able to efficiently initiate translation in their hosts. This interaction is proposed to occur through a guanosine residue that interacts with the cap-binding pocket of the protein in a similar way to the m7G cap (3). Control of eIF4E expression has important implications in plant susceptibility to viral infection and has been used to design more resistant crops (5-7). Its control is also important in mammalian systems: deregulated eIF4E has been implicated in many cancers including those of the breast (8), lung (9) and prostate (10), chronic human diseases such as diabetes (11), and developmental disorders such as autism (12). As eIF4E structure and function are highly conserved across kingdoms, and characterizing novel interactions with this protein have potential use in disease control, we sought to characterize this element in mammalian systems . We optimized translation in rabbit reticulocyte lysate and HeLa lysate for salt, RNA concentration and incubation time (Aim 1), compared the translation efficiency of reporter RNAs containing PTEs of 9 different viruses in lysates and whole cells (Aim 2), and investigated the ability of the PTE to act as an inhibitor (Aim 3). 1 CHAPTER 1. GENERAL INTRODUCTION: A REVIEW OF THE LITERATURE Historical Perspective In 1958, Sir Francis Crick coined the “Central Dogma of Molecular Biology” which revolutionized the thinking of the field (13). For the first time, it explicitly stated the way in which biological information flowed from nucleic acids to proteins, solidifying the assumptions that scientists had held while discussing the theories for protein expression from genes (14). It stated: “The central dogma of molecular biology deals with the detailed residue-by-residue transfer of sequential information. It states that such information cannot be transferred from protein to either protein or nucleic acid” (14). At this time, the fields of translation and its control were in their early years. In the 1950s, the field progressed rapidly: it was accepted that proteins were composed of unique combinations of 20 naturally occurring amino acids that were fastened together in an ordered fashion (13). The first protein sequences were published by Sanger and Tuppy, and radioisotopes allowed tracking of the fate of certain amino acids (15-19). In 1951, the first cell-free translation extracts were produced from rat livers, and it was discovered that both GTP and ATP were consumed in the translation reaction (20). In the same time period, protein synthesis was determined to occur rapidly at the ribosome, however, it was not entirely clear if the process required a template (15,21-23). Adaptor RNA, identified as tRNA was discovered and characterized in the late 1950s (24). In 1961, Jacob and
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