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Drosophila Melanogaster Dis3 Is a Dynamic Endo- and 3’Æ5’ DROSOPHILA MELANOGASTER DIS3 IS A DYNAMIC ENDO- AND 3’Æ5’ EXORIBONUCLEASE By MEGAN CHRISTINE MAMOLEN Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation advisor: Erik D. Andrulis, Ph.D. Department of Molecular Biology and Microbiology CASE WESTERN RESERVE UNIVERSITY August, 2010 We hereby approve the thesis/dissertation of ___________________Megan Mamolen_____________________ candidate for the ____________Ph.D.________________degree *. (signed)_________________Dr. Jonathan Karn_________________ (chair of the committee) _____________________Dr. Peter Harte______________________ ___________________Dr. Alan Tartakoff_____________________ _____________________Dr. Erik Andrulis_____________________ (date) _______6-15-10________________ *We also certify that written approval has been obtained for any proprietary material contained therein. 2 Copyright © 2010 by Megan Christine Mamolen All rights reserved 3 This work is dedicated to my husband and best friend, Mike Smolko. Thank you for encouraging me to believe in myself. This is only the beginning of a wonderful journey together. 4 Table of Contents Table of Contents ................................................................................................................ 5 List of Tables .................................................................................................................... 10 List of Figures ................................................................................................................... 11 Acknowledgements ........................................................................................................... 14 List of Abbreviations ........................................................................................................ 16 Abstract ............................................................................................................................. 20 Chapter I: Introduction ....................................................................................................................... 21 I.A RNA turnover and human health ............................................................................ 22 I.B RNA metabolism in eukaryotes .............................................................................. 23 I.B.1 RNA expression: RNA production versus RNA turnover ................................... 23 I.B.2 RNA processing: RNA stability and maturation ................................................. 25 I.B.3 RNA turnover: RNA destabilization and degradation ......................................... 26 I.C Ribonucleases ......................................................................................................... 31 I.C.1 Physical features of RNases .................................................................................. 32 I.C.2 RNase mechanisms of action ............................................................................... 34 I.C.3 RNase product formation and substrate specificity .............................................. 39 I.D Dis3 ......................................................................................................................... 45 I.D.1a Dis3 in vivo: mitosis............................................................................................. 45 I.D.1b Dis3 in vivo: RNA processing and turnover ........................................................ 46 I.D.1c Dis3 in vivo: additional features .......................................................................... 48 I.D.2 Dis3 in vitro characteristics ................................................................................. 49 5 I.E Summary ................................................................................................................. 54 I.F Hypothesis ............................................................................................................... 54 Chapter II: Characterization of the Drosophila melanogaster Dis3 Ribonuclease ............................. 58 II.A Abstract ................................................................................................................ 59 II.B Introduction ........................................................................................................... 60 II.C Materials and Methods .......................................................................................... 62 II.C.1 Molecular cloning ............................................................................................... 63 II.C.2 Expression and purification of recombinant proteins ......................................... 63 II.C.3 Preparation of RNA substrates ........................................................................... 64 II.C.4 Ribonuclease activity assays .............................................................................. 64 II.C.5 Quantification of RNase activity ........................................................................ 65 II.C.6 Dis3 immunoprecipitation .................................................................................. 65 II.D Results ................................................................................................................... 66 II.D.1 Recombinant dDis3 is active in vitro ................................................................. 66 II.D.2 MBP-dDis3 RNase activity requires monovalent and divalent cations ............. 66 II.D.3 MBP-dDis3 activity is not affected by non-ionic reaction conditions ............... 74 II.D.4 MBP-dDis3 RNase activity is not sequence specific ......................................... 77 II.D.5 dDis3 likely associates with exosome proteins via ionic and hydrophobic interactions ........................................................................................................................ 83 II.E Discussion .............................................................................................................. 85 II.E.1 Drosophila melanogaster Dis3 is a functional ribonuclease in vitro ................. 85 II.E.2 dDis3 Mg2+ requirements point to a metal-ion catalyzed reaction mechanism .. 87 6 II.E.3 dDis3 product formation may depend on substrate identity ............................... 88 II.E.4 dDis3 stably associates with core exosome proteins and exosome co-factors ... 90 II.E.5 Conclusions ......................................................................................................... 91 II.F Funding .................................................................................................................. 91 Chapter III: Drosophila melanogaster Dis3 N-terminal domains are required for ribonuclease activities, subcellular localization, and exosome interactions .......................................... 92 III.A Abstract ................................................................................................................ 93 III.B Introduction .......................................................................................................... 94 III.C Materials and Methods ......................................................................................... 97 III.C.1 Molecular cloning ............................................................................................. 97 III.C.2 Purification of recombinant proteins ................................................................. 97 III.C.3 Preparation of RNA substrates .......................................................................... 98 III.C.4 Ribonuclease activity assays ............................................................................. 98 III.C.5 Quantification of RNase activity ....................................................................... 99 III.C.6 Cell culture ........................................................................................................ 99 III.C.7 Immunofluorescence, immunoprecipitation, and western blotting ................... 99 III.C.8 Cell fractionation and isolation of mitochondria ............................................. 100 III.D Results ................................................................................................................ 101 III.D.1 MBP-dDis3 is active on linear RNAs of varying sequences .......................... 101 III.D.2 The dDis3 N-terminus harbors an endoribonuclease activity ......................... 101 III.D.3 dDis3 N-terminal domains are required for nuclear localization .................... 125 7 III.D.4 dDis3 localizes to mitochondria via an N-terminal mitochondria targeting sequence .......................................................................................................................... 131 III.D.5 dDis3 N-terminal domains are required for interactions with core exosome proteins and exosome co-factors ..................................................................................... 136 III.E Discussion........................................................................................................... 140 III.E.1 Dis3 N-terminal endoribonuclease activity is conserved in metazoans .......... 140 III.E.2 The dDis3 N-terminus is important for subcellular localization ..................... 142 III.E.3 The dDis3 N-terminus is responsible for interactions with core exosome proteins and exosome co-factors ..................................................................................... 144 III.E.4 Do dDis3 N-terminal domains link three different functions? .......................
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