Non-coding RNA 886 is a novel regulator of viral dsRNA sensors Open Access

Calderon, Brenda Milagros (Fall 2017)

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The innate immune response acts as a critical first line of defense against viral pathogens. Pattern recognition receptors on the cell surface and in the cytosol detect pathogen-associated molecular patterns and initiate signaling cascades to halt viral replication and establish an antiviral state. The double-stranded RNA-activated protein kinase (PKR) and the 2’-5’-oligoadenylate synthetases (OASes) sense dsRNA in the cytosol, a potent signal of viral infection, and limit viral replication through translational control. The central importance of PKR and OAS1 is highlighted by the abundance of viral strategies to inhibit their actions. The accurate discrimination of self from non-self is essential for normal cell function. Aberrant detection of self nucleic acids can lead to autoimmune disorders and improper activation of these sensors can contribute to human diseases including cancer. Thus, precise regulation of these sensors is required for proper cell function.

In this dissertation, the molecular mechanisms of PKR and OAS1 regulation by a cellular non-coding RNA, nc886, are investigated. Structural and biochemical studies reveal that nc886 adopts two stable conformations with stark differences in their functions towards PKR and OAS1. nc886 conformers differ structurally in their apical region with Conformer 1 adopting a unique tertiary structure. The presence of this structural motif confers high affinity PKR-binding and potent inhibition of PKR and its downstream activity on translation initiation factor 2. In contrast, Conformer 2 is a weak activator of PKR. Both conformers activate OAS1, but only Conformer 1 is capable of potent activation in vitro and in A549 cells. Functional analysis of various nc886 deletion variants reveals that PKR and OAS1 share overlapping, but distinct, requirements for nc886-mediated regulation and suggests they compete for binding. We present a model for nc886-mediated regulation of basal PKR activity in uninfected cells and propose that during infection nc886 activates OAS1 to stimulate the immune response. Finally, using growth arrest-specific 5 (Gas5), a long intergenic ncRNA, we demonstrate potential ncRNA-mediated regulation of immune sensors in response to diverse cell needs. Detailed knowledge of the cellular regulation of these sensors is essential for the development of therapeutic approaches to enhance or suppress the immune system.

Table of Contents

Chapter 1: Introduction1

1.1 The innate immune response to viral infections1

1.2 Nucleic acid sensing in innate immunity2

1.3 Discriminating self from non-self in innate immunity5

1.4 Translational control by PKR7

1.4.1 PKR cellular function7

1.4.2 Strategies viruses employ to evade activation of PKR9

1.5 Viral RNA degradation by the OAS/RNase L pathway10

1.5.1 OAS/RNase L cellular functions10

1.5.2 Strategies viruses employ to evade the OAS/RNase L pathway13

1.6 Non-coding RNA 88614

1.7 Research questions addressed by this work15

1.8 References22

Chapter 2: Human noncoding RNA 886 (nc886) adopts two structurally distinct conformers that are functionally opposing regulators of PKR32

2.1 Abstract32

2.2 Introduction33

2.3 Results35

2.3.1 nc886 RNA adopts two noninterconverting conformers with distinct stabilities 35

2.3.2 nc886 RNA conformers have opposing activities against PKR37

2.3.3 Differences in apical stem-loop structure distinguish the two nc886 RNA conformers38

2.3.4 The unique Conformer 1 apical stem-loop structure is critical for PKR repression40

2.4 Discussion43

2.5 Materials and Methods46

2.5.1 RNA in vitro transcription and purification46

2.5.2 PKR and eIF2α protein expression and purification47

2.5.3 RNA UV thermal melting analysis48

2.5.4 Electrophoretic mobility shift assays (EMSA)48

2.5.5 PKR inhibition assays49

2.5.6 PKR activation assays49

2.5.7 Selective 2’-hydroxyl acylation analyzed by primer extension (SHAPE)50

2.6 Acknowledgements51

2.7 References61

Chapter 3: Activation of the OAS/RNase L pathway by a human cellular non-coding RNA64

3.1 Abstract64

3.2 Introduction65

3.3 Results and Discussion67

3.3.1 OAS1 is potently activated by a single nc886 RNA conformer67

3.3.2 nc886 Conformer 1 activates the OAS/RNase L pathway in human A549 cells68

3.3.3 The nc886 terminal stem is dispensable for activation of OAS168

3.3.4 OAS1 activation requires an intact apical stem-loop70

3.4 Conclusions71

3.5 Materials and Methods72

3.5.1 RNA in vitro transcription and purification72

3.5.2 OAS1 expression and purification73

3.5.3 In vitro chromogenic assay of OAS1 activity73

3.5.4 OAS/RNase L activation in A549 cells74

3.5.5 RNA UV thermal melting analysis75

3.5.6 Selective 2’-hydroxyl acylation analyzed by primer extension (SHAPE)75

3.6 Acknowledgements76

3.6 References85

Chapter 4: Conclusion88

4.1 Structural differences in nc886 conformers have functional consequences89

4.2 PKR and OAS1 interact overlapping segments of nc886 RNA92

4.3 Gas5 lincRNA activates PKR94

4.4 Roles for nucleic acid sensors outside of immunity95

4.5 References100

Appendix: Gas5 lincRNA is a novel, cellular non-coding RNA activator of PKR102

A.1 Abstract102

A.2 Introduction102

A.3 Results104

A.3.1 Gas5 activates PKR in vitro104

A.3.2 Gas5 induces PKR T446 phosphorylation in HEK293T cells105

A.3.3 PKR recognition of Gas5 is length but not sequence dependent106

A.4 Discussion106

A.5 Materials and Methods108

A.5.1 RNA in vitro transcription and purification109

A.5.2 PKR protein expression and purification109

A.5.3 eIF2α protein expression and purification109

A.5.4 PKR activation assays110

A.5.5 Cell lines, transfections and immunoblotting110

A.6 Acknowledgements111

A.7 References116

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