2’-5’-oligoadenylate synthetase protein activation by cellular and viral non-coding RNAs Restricted; Files Only

Abstract

2’-5’-oligoadenylate synthetase protein activation by cellular and viral non-coding RNAs

By Alejandro Oviedo

The 2’-5’ oligoadenylate synthetase (OAS) family of innate immune sensors recognize cytosolic double-stranded RNA (dsRNA) to initiate an antiviral state through the production of 2’-5’ linked oligoadenylates that activate RNase L. Despite having a shared output, the three catalytically active OAS enzymes differ in their domain architecture, RNA preferences, and cellular distribution. The RNA features that drive OAS activation have been characterized almost entirely for OAS1 using minimal dsRNA substrates, leaving it unclear how these features translate to highly structured viral RNAs or whether these features extend to other family members, including the largest form, OAS3. This dissertation presents studies to address both of these gaps in current knowledge. First, I identify and characterize the structured RNA region within the SARS-CoV-2 5’-structural elements that drives potent OAS1 activation. Through systemic truncation of individual stem-loop and SHAPE-MaP structure probing, I identify SL1-4b as the minimal activating fragment. Based on these findings, I develop a model in which SL4 serves as the primary OAS1 interaction site while adjacent stem-loops (SL1and SL3) and an unstructured 3’-flanking region cooperate to optimally present SL4 for activation. This work establishes that a potent OAS1 activator within a complex viral RNA is not a canonical RNA helical (stem-loop) structure, but a larger assembly in which multiple RNA elements cooperate. Second, I focused on efforts toward a first structure of OAS3 and defining its mechanism of activation by the short cellular non-coding RNA nc886. I show that one nc886 conformer (conformer 1) is a potent activator of OAS3 despite being shorter than 50 bp, and thus below the previously established length requirement for OAS3 activation. Further, the relative activation by each nc886 conformer mirrors their ability to activate OAS1, establishing shared features of a structured RNA for activation of both OAS1 and OAS3. Efforts to determine the OAS-nc886 structure by cryo-EM did not yield a high-resolution model as the resulting reconstructions were only of low-resolution and thus unsuitable for confident interpretation. However, initial maps and AlphaFold3 modeling enabled initial predictions and should guide future efforts toward high-resolution experimental structures of OAS3-RNA complexes. Collectively, these findings argue that higher-order RNA structure is a recurring feature of RNAs that activate the OAS proteins and that known OAS1 activation enhancers may also extend to other OAS family members. 

Table of Contents

Table of Contents

Introduction ............................................................................................................ 1

The innate immune system ............................................................................................... 2

Nucleic acid sensing by the innate immune system ........................................................... 2

OAS2 ................................................................................................................................................. 9

OAS3 ............................................................................................................................................... 10

RNase L ........................................................................................................................................... 11

Detection of viruses by OAS proteins ............................................................................... 11

Flaviviridae ...................................................................................................................................... 12

Picornaviridae .................................................................................................................................. 13

Togaviridae ...................................................................................................................................... 13

Coronaviridae .................................................................................................................................. 14

RNA sequences and structures that promote OAS activation ............................................ 15

Research Goals .............................................................................................................. 18

Chapter 2 ......................................................................................................................................... 18

Chapter 3 ......................................................................................................................................... 18

References ..................................................................................................................... 20

SARS-CoV-2 5’-UTR stem-loops activate the antiviral protein oligoadenylate

synthetase 1 (OAS1) ............................................................................................... 26

Abstract ......................................................................................................................... 27

Introduction ................................................................................................................... 27

Results ........................................................................................................................... 29

The SARS-CoV-2 5’-SE RNA strongly activates the OAS family proteins. .............................................. 29

An extended region of the SARS-CoV-2 5’-SE is required for full OAS1 activation. ................................ 31

SL4b does not form a stable SL but may alter the SL1-4 RNA structure in a manner necessary for full

OAS1 activation. .............................................................................................................................. 34

SL1 and SL3 are needed for full activation of OAS1 by the SL1-4b RNA. ............................................... 37

SHAPE-MaP identification of OAS1-induced changes in SL1-4b. ......................................................... 38

Discussion ..................................................................................................................... 40

Experimental Procedures ................................................................................................ 42

RNA Synthesis .................................................................................................................................. 42

OAS1 protein expression and purification .......................................................................................... 43

In vitro OAS1 activation assay ........................................................................................................... 44

In-cell OAS/RNase L assay in A549 cells ............................................................................................ 44

Immunoblotting ............................................................................................................................... 45

2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) .................. 45

Data availability .............................................................................................................. 46

SUPPLEMENTAL FIGURES ................................................................................................ 48

SUPPLEMENTAL TABLES .................................................................................................. 54

References ..................................................................................................................... 56

The cellular non-coding RNA 886 (nc886) activates OAS3 ......................................... 60

Abstract ......................................................................................................................... 61

Introduction ................................................................................................................... 61

Results ........................................................................................................................... 63

Discussion ..................................................................................................................... 67

Methods ......................................................................................................................... 69

RNA Synthesis .................................................................................................................................. 69

OAS1 protein expression and purification .......................................................................................... 69

OAS3 protein expression and purification .......................................................................................... 70

In vitro OAS activation assay ............................................................................................................. 71

Electron microscopy grid preparation and negative staining ............................................................... 71

Specimen preparation and cryo-EM image collection and analysis ..................................................... 72

References ..................................................................................................................... 74

Discussion ............................................................................................................. 76

Summary of the Current Work ......................................................................................... 77

OAS1 recognition of SARS-CoV-2 ..................................................................................... 79

Determine OAS3-nc886 interactions ................................................................................ 80

OAS1 activation enhancers remain largely untested on OAS2 and OAS3 ............................ 82

nc886 as an endogenous activator of OAS1 and OAS3 ....................................................... 83

Final remarks .................................................................................................................. 84

References ..................................................................................................................... 85

Additional studies of nc886 structure and OAS interaction ....................................... 86

APPENDIX: Additional studies of nc886 structure and OAS interaction .............................. 87

Identification of sequences necessary for formation of the nc886 Conformer 1 AS tertiary structure. ... 87

Determine the first high-resolution structure of an OAS1-nc886 complex. .......................................... 88

Detection of nc886 in A549 and HeLa cells ........................................................................................ 89

In-cell SHAPE of nc886. .................................................................................................................... 90

References ..................................................................................................................... 92

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Biochemistry, Cell & Developmental Biology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Chemistry, Biochemistry Biology, Molecular
Keyword	RNA OAS
Committee Chair / Thesis Advisor	Graeme Conn, Emory University
Committee Members	Daniel Reines, Emory University Christine Dunham, Emory University Anita Corbett, Emory University Homa Ghalei, Emory University

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