Defining the RNA signatures detected by the innate immune sensor 2’-5’-oligoadenylate synthetase 1 (OAS1) 公开
Schwartz, Samantha (Spring 2021)
Abstract
The innate immune system is a broad set of critical intracellular and extracellular processes that limit pathogen infectivity. To provide its essential first line of defenses, the innate immune system must identify and respond to foreign molecules. The 2’-5’-oligoadenylate synthetase (OAS) family of enzymes are important innate immune sensors of cytosolic double-stranded RNA (dsRNA), a potent signal and hallmark of viral infection. Defective or misregulated innate immune activity can cause increased persistence of and susceptibility to viral infection and human diseases, such as interferonopathies. My dissertation research investigates how specific molecular signatures within dsRNA molecules regulate OAS1 activity.
Structural studies previously revealed that dsRNA binding induces allosteric changes in OAS1 that reorganize its catalytic site and drive synthesis of 2’-5’-oligoadenylates from ATP. However, we still understand relatively little about how specific features of the dsRNA contribute to the level of OAS1 activation. I used a “simple” 18 bp dsRNA containing two copies of a previously identified OAS1 activation consensus sequence (WWN9WG, where W is A or U, and N is any nucleotide) to identify and assess the contributions of specific dsRNA features required for potent OAS1 activation. Biochemical studies coupled with cell-based assays and computational modeling reveal that the 18 bp dsRNA contains two competing OAS1 binding sites with remarkably different capacities to activate the protein in a context-dependent manner. The dsRNA binding register was dictated by the position of the consensus sequence(s) and only when optimally positioned was potency of other RNA activating motifs observed. A new role was also identified for the non-conserved (N9) nucleotides of the activation consensus in defining the dsRNA shape and flexibility important for OAS1 binding and recognition. The results indicate that OAS1 is unexpectedly sensitive to sequence, but also that the relative placement and organization of activating motifs is as critical as the sequences themselves. These studies define the molecular mechanism(s) that control OAS1 regulation by dsRNA and may reveal insight into viral evasion of the OAS1/RNase L pathway or avenues to new effective therapeutic strategies.
Table of Contents
Chapter 1: Introduction ...................................................................................................1
Innate immunity............................................................................................................2
Viral nucleic acid sensing.............................................................................................2
Viral evasion strategies for the OAS/RNase L pathway...............................................7
Roles for the OAS/RNase L pathway in normal cell function.......................................9
OAS1 consensus sequences and activating motifs...................................................10
Research goals..........................................................................................................14
References.................................................................................................................18
Chapter 2: RNA regulation of the antiviral protein 2’-5’-oligoadenylate synthetase ......................................................................................................................26
Abstract ......................................................................................................................27
Introduction ................................................................................................................28
Sidebar: The innate immune system ...................................................................29
Sidebar: Alternative splicing expands human OAS protein diversity ...................32
Molecular basis of OAS1-3 regulation by dsRNA ......................................................33
OAS1 regulation by short dsRNAs.......................................................................33
Basis of long dsRNA sensing by OAS3 ...............................................................37
OAS2 is activated by dsRNA of intermediate length............................................38
Role of RNA sequence and structure in OAS activation............................................39
RNA sequence specificity in OAS activation........................................................40
OAS1 “consensus” sequences.......................................................................40
Sequence and structural motifs: 3’-ssPy motif and nc886 RNA.....................41
Coding and non-coding viral RNAs containing dsRNA regions ...........................46
Conclusions ...............................................................................................................48
Acknowledgments ......................................................................................................51
Funding ......................................................................................................................52
References.................................................................................................................52
Chapter 3: Human OAS1 activation is highly dependent on both RNA sequence and context of activating RNA motifs..........................................................................60
Abstract ......................................................................................................................61
Introduction ................................................................................................................62
Materials and Methods...............................................................................................65
OAS1 protein expression and purification............................................................65
Generating 18 bp dsRNA duplexes .....................................................................65
Chromogenic assay of OAS1 activity...................................................................66
OAS1/RNase L activation in A549 cells...............................................................67
OAS1-dsRNA 4-thiouridine (4-thioU) crosslinking ...............................................67
Results .......................................................................................................................68
The 3’-ssPy motif enhances OAS1 activation only when appended to the top strand of the 18 bp dsRNA................................................................................68
Short dsRNAs activate the OAS1/RNase L pathway in human A549 cells mirroring their capacity to activate OAS1 in vitro ..............................................71
The 3’-ssPy placement can alter apparent dsRNA affinity and maximal OAS1 activation ...........................................................................................................73
Altering the distance between the 3’-ssPy motif and the consensus sequence impacts its ability to enhance OAS1 activation .................................................73
OAS1 activation consensus sequence nucleotide identities (WW/WG) and their placement within a dsRNA are important for OAS1 activation..........................77
Discussion..................................................................................................................79
Acknowledgments ......................................................................................................85
Funding ......................................................................................................................85
References.................................................................................................................85
Chapter 4: Basis for sequence-dependent activation of the innate immune dsRNA sensor oligoadenylate synthetase 1 ............................................................................91
Abstract ......................................................................................................................92
Introduction ................................................................................................................93
Materials and Methods...............................................................................................96
OAS1 protein expression and purification............................................................96
Generating 18 bp dsRNA duplexes .....................................................................96
Chromogenic assay of OAS1 activity...................................................................97
OAS1/RNase L activation in A549 cells...............................................................97
Immunoblotting ....................................................................................................98
RNA UV melting analysis.....................................................................................99
Thermal difference spectra (TDS)........................................................................99
dsRNA molecular dynamics (MD) simulations...................................................100
Results .....................................................................................................................101
A-to-I “editing” does not prevent OAS1 activation..............................................101
OAS1 activation is enhanced by inosine and mismatches in the center of short dsRNAs ..............................................................................................................104
Stabilization with G-C substitutions can also enhance OAS1 activation by short dsRNAs ..............................................................................................................106
Differences in dsRNA structure correlate with their capacity to activate OAS1 .108
MD simulation reveals a role for helical dynamics in OAS1 activation ..............112
Specific base pair changes pre-dispose helical parameters to favor the short dsRNA adopting an OAS1-bound-like conformation..........................................116
Additive effects of distinct helical features on OAS1 activation .........................117
Short dsRNA-mediated OAS1/RNase L activation in human cells mirrors their activity in vitro ....................................................................................................118
Discussion................................................................................................................120
Acknowledgments ....................................................................................................123
Funding ....................................................................................................................123
References...............................................................................................................123
Chapter 5: OAS1 activation is determined by competing activating and non- activating binding sites in double-stranded RNA.....................................................135
Introduction ..............................................................................................................136
Materials and Methods.............................................................................................138
OAS1 protein expression and purification..........................................................138
Generating 18 bp dsRNA duplexes ...................................................................138
Chromogenic assay of OAS1 activity.................................................................139
OAS1/RNase L activation in A549 cells.............................................................140
OAS1-dsRNA 4-thiouridine (4-thioU) crosslinking .............................................140
Results .....................................................................................................................141
Mutation of the conserved guanine nucleotide (WWN9WG) reveals that two activating consensus sequences within the same dsRNA are not equivalent 141
OAS/RNase L pathway activation in human A549 cells mirrors OAS1 activity in vitro .................................................................................................................145
Consensus 1, but not Consensus 2, requires both the guanine and Watson-Crick base pairing for potent OAS1 activation .........................................................146
Consensus sequence changes impact both dsRNA affinity (Kapp) and maximal OAS1 activation (Vmax) ....................................................................................149
Discussion................................................................................................................149
References...............................................................................................................153
Chapter 6: Discussion.................................................................................................155
Define the molecular mechanism(s) of action for enhancement of OAS1 activation by RNA sequence and structural motifs ....................................................................159
Expand the model dsRNA to test the action of the OAS1 activation consensus sequence and other activating motifs in the context of longer or more structured RNA ......................................................................................................................161
Identify the impact (if any) these OAS1 activation sequences have on other OAS family members, OAS2 and OAS3 .......................................................................161
Pathway crosstalk and the additional role RNAs could play in regulating multiple arms of the innate immune system.......................................................................162
Determine the molecular basis of dsRNA-independent OAS1 activation conferred by clinical mutations with gain-of-function phenotypes..............................................163
Develop novel OAS1 inhibitors (and activators) for therapeutic implementation.....164
Final remarks ...........................................................................................................167
References...............................................................................................................167
TABLE OF FIGURES
Chapter 1
Figure 1. Overview of the innate immune system: cytosolic dsRNA receptors...........4
Figure 2. A-to-I editing by ADAR1 is proposed to reduce self-activation of innate
immune response pathways .....................................................................................6
Figure 3. Viral evasion strategies to circumvent the OAS/RNase L pathway .............8
Figure 4. dsRNA binding induces conformational changes required for 2-5A synthesis by OAS1 .................................................................................................11
Figure 5. Overview of research objectives................................................................15
Chapter 2
Graphical/Visual Abstract and Caption .................................................................28
Figure 1. Overview of the OAS/RNase L pathway ....................................................30
Figure 2. Conformational changes induced by dsRNA binding promote synthesis of 2-5A by OAS1.........................................................................................................34
Figure 3. Double-stranded and structured RNA activators of OAS...........................42
Figure 4. Model for enhancement of OAS1 activation by RNA sequence and structural motifs ......................................................................................................45
Figure 5. Summary of OAS protein activation by RNA .............................................50
Chapter 3
Figure 1. The OAS1/RNase L pathway and 18 bp dsRNA design............................63
Figure 2. The 3’-ssPy motif impacts OAS1 activity only when appended to the top strand ......................................................................................................................70
Figure 3. OAS/RNase L pathway activation in A549 cells by the short dsRNAs correlates with their ability to activate OAS1 in vitro...............................................72
Figure 4. The 3’-ssPy motif differentially alters the kinetics of OAS1 activation when appended to the top or bottom strand.....................................................................74
Figure 5. The relative context of the 3’-ssPy motif and consensus sequence is important for OAS1 activation.................................................................................76
Figure 6. The OAS1 activation consensus sequence (WWN9WG) and its placement within dsRNA are also important for OAS1 activation ............................................78
Figure 7. Models of OAS1 interactions with 18 bp dsRNA variants and the 3’-ssPy motif ........................................................................................................................82
Chapter 4
Figure 1. IU base pairs do not diminish OAS1 activation by a short dsRNA.........102
Figure 2. OAS1 activation is potentiated by inosine substitutions and mutations that introduce non-canonical Watson Crick base pairing................................................105
Figure 3. Homopolymer sequence strongly influences OAS1 activation ................107
Figure 4. dsRNA helix stabilizing G-C base pairs enhance OAS1 activation..........108
Figure 5. Thermal difference spectra (TDS) identify structural changes in dsRNA that correlate with increased OAS1 activation ................................................................110
Figure 6. Increased dsRNA dynamics and specific helical features underpin enhanced OAS1 activation by short dsRNAs ..........................................................114
Figure 7. Sequence changes resulting of enhancement of OAS1 distinct via distinct molecular mechanisms have additive activity ..........................................................118
Figure 8. Activation of the OAS1/RNase L pathway by WWN9WG dsRNA variants mirrors their capacity to activate OAS1 in vitro ........................................................120
Figure S1. Native gel analysis showing purity of ssRNAs and stable formation of each dsRNA.............................................................................................................128
Figure S2. Workflow for determining thermal difference spectra (TDS)..................129
Figure S3. The poly(rG:rC) homopolymer is a poor activator even at high
concentrations..........................................................................................................129
Figure S4. MD analysis of benchmark sequence and dsRNA RMSD.....................130
Figure S5. Mismatch base pairings (I•U and A•C) cause major alterations helical structure ...................................................................................................................131
Figure S6. Analysis of helical and base pair/ base step parameters for GC2 and GC3 dsRNAs ...........................................................................................................132
Figure S7. Analysis of helical and base pair/ base step parameters for AC1-GC2 dsRNA......................................................................................................................133
Chapter 5
Figure 1. A short 18 bp dsRNA can contain two OAS1 activation consensus
sequences (WWN9WG) with vastly different capacities to activate OAS1............142
Figure 2. CS1-AC (non-activator) and CS2-AC (hyper-activator) form stable OAS1- dsRNA complexes ................................................................................................144
Figure 3. OAS/RNase L pathway activation in A549 cells mirrors OAS1 activity by the short dsRNAs in vitro ......................................................................................145
Figure 4. Consensus 1 requires both the conserved guanine and Watson-Crick base pairing for potent OAS1 activation ........................................................................147
Figure 5. OAS1 activation does not require the conserved guanine in Consensus 2, and in specific contexts, altering the base content in this location significantly improves OAS1 activity.........................................................................................148
Figure 6. Changes in the two OAS1 activation consensus sequences dramatically alter apparent dsRNA affinity and maximal OAS1 activation................................150
Chapter 6
Figure 1. Overview of main research findings.........................................................159
Figure 2. dsRNA-independent OAS1 gain-of-function activity is favored by allosteric loss of structural constraints .................................................................................165
LIST OF TABLES
Chapter 3
Table 1. Summary of OAS1 activation by short dsRNAs in vitro and A549 cells ......90
Chapter 4
Table 1. Summary of short dsRNA stability and OAS1 activation...........................127
Table S1. Summary of dsRNA UV melting analysis................................................134
Chapter 5
Table 1. Summary of OAS1-dsRNA kinetic parameters..........................................154
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