RNA RECOGNITION BY THE PATTERN RECOGNITION RECEPTOR RIG-I: ROLES OF RNA BINDING, MULTIMERIZATION, AND RNA-DEPENDENT ATPASE ACTIVITY By ELIZABETH E. DELANEY 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 August 2014 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve this thesis/dissertation of Elizabeth E. DeLaney . Candidate for the Doctor of Philososphy degree* (signed) David Samols . (chair of the committee) Eckhard Jankowsky . Derek Abbott . Blanton Tolbert . Jonatha Gott . (date) March 28, 2014 . *We also certify that written approval has been obtained for any proprietary material contained therein. ii Table of Contents List of Tables…………………………………………………………………………….ix List of Figures…………………………………………………………………………….x Acknowledgements>……………………………………………………………………xiv List of Abbreviations……………………………………………………………….…..xvi Abstract…………………………………………………………………………………xix Chapter 1: Pathogen detection by the innate immune system 1.1 Innate immunity: detection of conserved molecular patterns…………………...1 1.2 Types of PRRs……………………………………………………………………2 1.2.1 Toll-like receptors……………………………………………………….2 1.2.2 C-type lectin receptors………………………………………………......5 1.2.3 Nod-like receptors………………………………………………………7 1.2.4 RIG-I-like receptors……………………………………………………..9 1.2.4.1 Identification and function of RIGI……………………………...10 1.2.4.2 Identification and function of MDA5 and LGP2………………...11 1.3 RIG-I signaling pathway…………...…………………………………………...13 iii 1.4 RIG-I and MDA5 activate antiviral signaling in response to distinct viruses…17 1.4.1 RIG-I recognizes a diverse range of viruses………………………….17 1.4.2 MDA5 is primarily activated by picornaviruses……………………...20 1.5 RIG-I domain structure and function…………………………………………..20 1.5.1 Overall RIG-I domain structure………………………………………20 1.5.2 CARD domains in RIG-I are necessary for signal transduction………23 1.5.2.1 Structure and function of RIG-I CARD domains………………..23 1.5.2.2 Ubiquitination of the CARD domains regulates RIG-I signal transduction………………………………………………………26 1.5.3 RIG-I helicase/ATPase domain………………………………………..30 1.5.4 RIG-I CTD functions in ligand binding and multimerization…………37 1.5.5 Molecular mechanism of RIG-I substrate recognition…………….…..41 1.6 RIG-I recognizes multiple structural features of viral RNA……………………45 Chapter 2: Development of L21 ribozyme as a method to generate RIG-I RNA substrates……………………………………………………………………51 2.1 Rationale for using the L-21 ribozyme for producing RIG-I substrates in vitro.....................................................................................................................51 2.2 Characterization of L-21 ribozyme activity on RIG-I substrates……….……...54 2.3 Discussion……………………………………………………………………..60 iv Chapter 3: RIG-I tightly binds dsRNA………………………………………………....61 3.1 Introduction……………………………………………………………………61 3.2 Results…………………………………………………………………………62 3.2.1 Purification of wild-type and mutant RIG-I………………………………62 3.2.2 RIG-I binds ssRNA with low affinity despite presence of a 5’-triphosphate……………………………………………………………63 3.2.3 5’-triphosphate does not impact RIG-I binding to dsRNA……………….64 3.2.4 RIG-I binds dsRNA tightly regardless of duplex length………………….68 3.2.5 RIG-I binding to RNA duplexes at least 16 bp involves two species or RNA-protein complexes………………………………………..…………70 3.2.6 RIG-I multimerization is dependent upon RNA duplex length…….…....72 3.2.7 RIG-I deletion mutant demonstrate major RNA binding site is the CTD..77 3.3 Discussion……………………………………………………………………...84 3.3.1 RNA 5’-end structure has no significant effect on RIG-I RNA binding….84 3.3.2 RIG-I RIG-I binds dsRNA cooperatively and multimerization is dependent on duplex length…………………………………………………………..87 3.3.3 Major RNA binding site is in the RIG-I CTD…………………………….88 Chapter 4: RIG-I ATPase activity recognizes the presence of dsRNA……..………….90 v 4.1 Introduction………………………………………………………..…………90 4.2 Results…………………………………………..……………………………91 4.2.1 Nucleotide has no effect on RIG-I-RNA affinity, but promotes complex dissociation………………………………………………….…………………91 4.2.2 ATP enhances dissociation of RIG-I from RNA………………………..96 4.2.3 RIG-I RNA-dependent ATPase activity is independent of RNA duplex length…………………………………………………………………...100 4.3 Discussion…………………………………………………………………….102 Chapter 5: RIG-I proofreads RNA duplexes for blunt ends with its ATPase activity…106 5.1 Introduction…………………………………………………………………...106 5.2 Results…………………………………………………………………………107 5.2.1 RIG-I efficiently binds RNA duplexes with blocked ends………………107 5.2.2 RIG-I ATPase activity proofreads RNA duplexes for blunt ends……….109 5.3 Discussion…………………………………………………………………….112 5.3.1 RIG-I RNA-dependent ATPase activity identifies blunt duplex ends…...112 5.3.2 Model of RIG-I substrate recognition……………………………………113 Chapter 6: Future directions……………………………………………………………116 vi 6.1 Introduction……………………………………………………………………...116 6.2 Effects of RNA 5’-end structure on RIG-I RNA recognition in vivo……….117 6.3 Activation of antiviral signaling in vivo by frayed end substrates…………..118 6.4 Effect of ubiquitination of RIG-I on RIG-I-RNA binding and ATP hydrolysis………………….............................................................................119 Chapter 7: Materials and Methods…………………………………………………….124 7.1 Materials………………………………………………………………………124 7.1.1 Plasmids…………………………………………………………………124 7.1.2 Proteins………………………………………………………………….126 7.1.3 Oligonucleotides………………………………………………………...127 7.1.4 Miscellaneous reagents………………………………………………….130 7.2 Methods………………………………………………….……………………131 7.2.1 Radiolabeling and gel purification of RNA and DNA oligonucleotides..131 7.2.2 Characterization of L-21 ribozyme activity on RIG-I substrates………..132 7.2.3 End-labeling of ssRNA with L-21 ribozyme…………………………….132 7.2.4 RIG-I binding under equilibrium conditions…………………………….133 7.2.5 Derivation of equations for coupled equilibrium………………………..134 vii 7.2.6 Dissociation kinetics of RIG-I-RNA complexes………………………...136 7.2.7 Glutaraldehyde crosslinking of RIG-I-RNA complexes…………………137 7.2.8 Measurement of RIG-I ATPase activity with thin layer chromatography.138 Bibliography……………………………………………………………………………139 viii List of Tables Table 1.1. Viruses recognized by RIG-I………………………………………………...19 Table 2.1. L-21 ribozyme RNA precursor substrate sequences…………………………55 Table 3.1. Wild-type RIG-I binding parameters calculated from coupled equilibrium…71 Table 3.2. Stimulation of RIG-I ATPase activity by 36 nt ssRNAs and 36 bp duplexes with and without a 5’-triphosphate……………………………………...…..79 Table 3.3. RIG-IΔCARD binding parameters calculated from coupled equilibrium...…82 Table 4.1. RIG-I binding to 36 bp duplex with a 5’-triphosphate in the presence of nucleotide…………………………………………………………………...92 Table 4.2. Off rates of RIG-I from indicated RNA duplexes in presence and absence of ATP…………………………………………………………………………..99 Table 5.1. Dissociation rate constants and kinetic parameters of RIG-I in presence of end-blocked RNA substrates………………………………………………111 ix List of Figures Figure 1.1. Schematic of the major classes of cellular PRRs……………………………4 Figure 1.2. Schematic of RIG-I domain structure……………………………………….10 Figure 1.3. Schematic of MDA5 and LGP2 domain structures…………………………13 Figure 1.4. Schematic of RLR signaling pathway………………………………………15 Figure 1.5. Sequence alignment of RIG-I, MDA5, and LGP2………………………….21 Figure 1.6. Schematic showing the Greek key motif present in CARD domains………24 Figure 1.7 Structure of MAVS CARD domain…………………………………………24 Figure 1.8 Crystal structure of duck RIG-I in the absence of RNA and NTP………….25 Figure 1.9. TRIM25 interacts with T55 in CARD1 to ubiquitinate K172 in CARD2…27 Figure 1.10. Schematic of RIG-I domain structure with residues ubiquitinated by Riplet………………………………………………………………………28 Figure 1.11. Schematic of RIG-I domain structure with residue important for interaction with TRIM25 shown and the interaction of the second CARD domain with K63-linked polyubiquitin chains…………………….……………………29 Figure 1.12. Schematics of conserved RNA helicase domain motifs…………………..32 Figure 1.13. Crystal structure of ligand bound human RIG-I………………………......37 Figure 1.14. Schematic and crystal structure of RIG-I CTD……………………………40 x Figure 1.15 RIG-I undergoes a conformational change upon RNA binding…...……….43 Figure 2.1. Tetrahymena ribozyme crystal structure and schematic of mechanism……54 Figure 2.2. L-21 ribozyme efficiently cleaves R13L21PBot precursor RNA………......57 Figure 2.3. L-21 ribozyme efficiently cleaves R13L21P and R10L21P precursor RNAs………………………………………………………………………..58 Figure 2.4. L-21 ribozyme efficiently cleaves and 84 nt precursor in the presence of GTP And analogs……………………………………….………………………59 Figure 3.1. Schematic of wild-type RIG-I and RIG-I deletion mutants……………….62 Figure 3.2. Purified WT and mutant RIG-I proteins……………………………….…..63 Figure 3.3. RIG-I binds ssRNA weakly, and 5’-triphosphate enhances binding affinity…………………………………………………………………….64 Figure 3.4. RIG-I binds a 36 bp dsRNA tightly, regardless of presence of a 5’-triphosphate……………………………………………………………..65 Figure 3.5. RIG-I binds a 10 bp dsRNA tightly, regardless of presence of a 5’-triphosphate..............................................................................................67 xi Figure 3.6. RIG-I binds a 13 bp dsRNA tightly, regardless of presence of a 5’-triphosphate...............................................................................................68 Figure 3.7. RIG-I binds dsRNA tightly