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Understanding the Molecular Mechanisms of The UNDERSTANDING THE MOLECULAR MECHANISMS OF THE RNA HELICASES DHX36 AND DDX41 by SUKANYA SRINIVASAN Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation advisor: Dr. Eckhard Jankowsky Department of Biochemistry CASE WESTERN RESERVE UNIVERSITY May 2020 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Sukanya Srinivasan Candidate for the Doctor of Philosophy degree* William C. Merrick (chair of the committee) Eckhard Jankowsky Derek J. Taylor Tsan Sam Xiao (date) March 18, 2020 *We also certify that written approval has been obtained for any proprietary material contained therein. Table of contents List of figures..................................................................................................................viii List of tables.....................................................................................................................xii Acknowledgment ............................................................................................................ xiii List of abbreviations ........................................................................................................xv Abstract ......................................................................................................................... xviii Chapter 1: General Introduction to SF2 RNA helicases ................................................1 1.1 Introduction to RNA helicases ................................................................................ 1 1.2 Classification of helicases ....................................................................................... 1 1.3 Structural architecture and conserved sequence motifs of SF2 RNA helicases ..... 3 1.3.1 Helicase core and its conserved sequence motifs ............................................ 3 1.3.2 Terminal accessory domains ........................................................................... 6 1.3.3 β-hairpin .......................................................................................................... 9 1.4 Distinct modes of duplex unwinding by SF2 RNA helicases ............................... 11 1.5 Biochemical activities & cellular functions of SF2 RNA helicases ..................... 13 1.5.1 Diverse cellular functions .............................................................................. 13 1.5.2 RNA unwinding ............................................................................................ 15 1.5.3 Protein displacement ..................................................................................... 15 1.5.4 Strand annealing ............................................................................................ 16 1.5.5 RNA structure conversion and chaperone activity ........................................ 16 1.5.6 Viral nucleic-acid and bacterial pathogen sensing ........................................ 17 1.6 Influence of cofactors on RNA helicase function ................................................. 17 i 1.7 Studies on DHX36 and DDX41 in this thesis ....................................................... 18 1.7.1 The DEAH/RHA helicase DHX36 ................................................................ 19 1.7.2 The DEAD-box protein DDX41 ................................................................... 19 Chapter 2: Introduction-The DEAH/RHA helicase is involved in the regulation of gene expression ................................................................................................................21 2.1 Gene regulation in eukaryotes .............................................................................. 21 2.1.1 Transcriptional regulation ............................................................................. 21 2.1.2 Post-transcriptional regulation ...................................................................... 23 2.2 Nucleic acid structures and gene regulation ........................................................ 26 2.2.1 Classical DNA/RNA structures and their functions ...................................... 26 2.2.2 G-quadruplexes ............................................................................................. 29 2.2.2.1 Genomic mapping of G-quadruplexes ................................................ 31 2.2.2.2 In vivo existence of G-quadruplexes ................................................... 33 2.2.2.3 Biological roles of G-quadruplexes .................................................... 33 2.2.2.4 G-quadruplex disease connections ...................................................... 40 2.2.2.5 G-Quadruplex drug targeting .............................................................. 42 2.3 NA binding proteins ............................................................................................. 43 2.3.1 General aspects of NA-binding proteins ...................................................... 44 2.3.2 G-quadruplex (GQ) interacting proteins ....................................................... 46 2.4 The DEAH/RHA helicase, DHX36 ...................................................................... 49 2.5 Biological roles of DHX36 ................................................................................... 49 2.5.1 Disease relevance .......................................................................................... 55 2.6 NA remodeling mechanism of DHX36 ................................................................ 57 ii 2.7 Structural basis for NA remodeling by DHX36 ................................................... 59 2.8 Structural and biochemical analyses of RNA remodeling activity of DHX36 .... 61 Chapter 3: Introduction-The DEAD-box helicase DDX41, a myelodysplastic syndrome implicated splicing factor ..............................................................................63 3.1 Conservation and structure of human DEAD-box protein DDX41 ...................... 63 3.2 Cellular Functions of DDX41 ............................................................................... 66 3.2.1 Abstrakt the drosophila homolog of DDX41 ................................................ 66 3.2.2 DDX41 in the spliceosomal complex ............................................................ 67 3.2.3 DDX41 in innate immune signaling .............................................................. 68 3.2.4 DDX41 and tumor development ................................................................... 73 3.2.5 DDX41 in post-transcriptional gene regulation ............................................ 74 3.3 Myelodysplastic syndrome ................................................................................... 74 3.3.1 Myelodysplastic syndrome associated protein factors .................................. 75 3.3.1.1 Splicing factors in myeloid neoplasms ............................................... 76 3.3.1.2 DDX41 in myeloid neoplasms ............................................................ 77 3.4 Cellular RNA targets of DDX41 and cellular consequences of DDX41 perturbations ............................................................................................................. 80 3.5 Biochemical characterization of DDX41 .............................................................. 82 Chapter 4: Function of auxiliary domains of the DEAH/RHA helicase DHX36 in RNA remodeling ...............................................................................................................83 4.1 Introduction ........................................................................................................... 83 4.2 Results ................................................................................................................... 83 4.2.1 Crystal structure of mouse DHX36 ............................................................... 83 iii 4.2.2 Remodeling of RNA duplexes and quadruplexes by mDHX36 .................... 87 4.2.3 The DSM promotes remodeling of RNA quadruplexes and duplexes and binding to RNA quadruplexes .............................................................................. 91 4.2.4 The OB-fold promotes binding and remodeling of quadruplex and duplex structures. .............................................................................................................. 95 4.2.5 The β-hairpin promotes binding and remodeling of quadruplex and duplex structures. .............................................................................................................. 98 4.3 Discussion ........................................................................................................... 103 Chapter 5: The nucleotide selectivity of DHX36 influences its RNA substrate selectivity and vice versa ...............................................................................................108 5.1 Introduction ......................................................................................................... 108 5.2 Results ................................................................................................................. 110 5.2.1 Nucleotide binding site of mouse DHX36 .................................................. 110 5.2.2 NTP-dependent remodeling of RNA duplexes and quadruplexes by mDHX36 ............................................................................................................................. 112 5.2.3 Physiological ATP levels inhibit mDHX36 remodeling of RNA duplexes but not quadruplexes ................................................................................................
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