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 ................................................................................................