The Multifaceted Functions of the Long Noncoding RNAs NEAT1 and GOMAFU are Regulated by the RNA-Binding Protein Quaking Restricted; Files Only

Abstract

Approximately 2% of the human genome encodes proteins while the rest comprise the noncoding genome that is highly transcribed. Within the noncoding transcriptome, small noncoding RNAs (e.g., microRNAs, snRNAs, snoRNAs, etc.) are highly conserved, whereas long noncoding RNAs (lncRNAs), including linear and circular forms, are poorly conserved across species. Numerous lncRNAs are highly enriched in the human brain but dysregulated in neurodevelopmental and neuropsychiatric diseases, as well as brain tumors. However, the molecular mechanisms regulating lncRNA expression in normal and diseased brains remain largely elusive. A candidate for regulating lncRNAs is the RNA-binding protein (RBP) Quaking I (QKI). QKI is abundantly expressed in neural stem cells and known to control neuron-glia lineage development. The QKI gene produces three protein isoforms with distinct nuclear-cytoplasmic distribution. The nuclear isoform QKI-5 governs alternative splicing of coding transcripts and regulates the biogenesis of microRNAs and circular RNAs. In this work, we investigate the functions of the lncRNA Nuclear Paraspeckle Assembly Transcript 1 (NEAT1) in glioblastoma, as well as the lncRNA GOMAFU in schizophrenia. Moreover, we explore whether the RBP QKI regulates these two lncRNA. To analyze the regulation of NEAT1 lncRNA function in glioblastoma, we first provide evidence that the NEAT1 isoforms NEAT1_1 and NEAT1_2 are differentially expressed in human glioblastoma cells. We determined that deletion of the proximal polyadenylation site (PAS) of NEAT1 increases the production of NEAT1_2. To investigate the regulation of NEAT1 PAS usage, we identified a putative RNA-binding consensus sequence for the RBP QKI. We show knockdown of a nuclear form of QKI, QKI-5, results in alterations to the relative abundance of NEAT1 isoforms at steady-state. Moreover, we show changes in NEAT1 isoform steady-state levels alter the GBM transcriptome. Analysis of GOMAFU revealed distinct changes in transcript formation at steady-state. We observe GOMAFU steady-state levels increase upon loss of QKI-5 via CRISPR-Cas9 modifications in a human neural progenitor cell model, suggesting QKI-5 may suppress GOMAFU production. We show loss of QKI-5 leads to increased levels of GOMAFU, indicating QKI-5 suppresses GOMAFU formation and/or stability. Moreover, while GOMAFU is involved in organizing RBPs, we observe no changes in RBP localization upon loss of QKI-5. Altogether, these studies demonstrate the multifaceted roles of NEAT1 and GOMAFU and elucidate how QKI functions via distinct mechanisms to regulate lncRNAs. These studies provide a new understanding for how dysregulation of lncRNAs, involving RBP abnormalities, are important mechanisms underlying complex etiology of brain tumors and neuropsychiatric diseases.

Table of Contents

CHAPTER 1: INTRODUCTION

1.1 Long noncoding RNA: 2

1.1.1 An overview of noncoding RNAs: 2

1.1.2 Long noncoding RNA definition, classification, and function in gene regulation: 6

1.1.3 LncRNAs function to regulate gene expression: 9

1.1.4 The dysregulation of lncRNAs contributes to central nervous system disorders: 11

1.1.5 Dysregulation of lncRNAs impacts cancer progression: 14

1.2 The long noncoding RNA NEAT1: 20

1.2.1 Identification and characterization of the two NEAT1 isoforms: 20

1.2.2 NEAT1 is dysregulated in neurological diseases and disorders: 24

1.2.3 NEAT1 is implicated in numerous types of cancer: 25

1.2.4 NEAT1 in glioma and glioblastoma multiforme: 28

1.3 Nuclear Paraspeckles: 31

1.3.1 Identification of the proteins essential for paraspeckles: 31

1.3.2 The formation and structure of paraspeckles: 34

1.3.3 The molecular functions of paraspeckles and implications in diseases: 39

1.4 The long noncoding RNA GOMAFU and its potential role as a risk factor for Schizophrenia: 40

1.4.1 Schizophrenia, candidate genes, and alternative splicing during neurodevelopment: 40

1.4.2 Discovery of GOMAFU and disease associated genetic variants: 43

1.4.3 The lncRNA GOMAFU in neuronal development and neurophysiological function: 48

1.4.4 Dysregulation of human GOMAFU is a potential risk factor for Schizophrenia: 50

1.4.5 Mechanisms for GOMAFU to regulate the downstream molecular pathways in Schizophrenia: 52

1.5 The functional importance of the selective RNA-binding protein Quaking in RNA regulation: 53

1.5.1 Discovery of the Quaking gene and proteins: 53

1.5.2 The function and mechanism of QKI as an RNA-binding protein: 60

1.5.3 The role of QKI in diseases: 61

1.6 Unknown questions and scope of this dissertation: 65

CHAPTER 2: ISOFORM BALANCE OF THE LONG NONCODING RNA NEAT1 IS REGULATED BY THE RNA-BINDING PROTEIN QKI, GOVERNS THE GLIOMA TRANSCRIPTOME, AND IMPACTS CELL MIGRATION

2.1 Abstract: 70

2.2 Introduction: 71

2.3 Results: 74

2.3.1 Quantification of the steady-state levels of NEAT1 isoforms revealed differential dysregulation in human GBM GSCs: 74

2.3.2 Deletion of the NEAT1 PAS results in diminished NEAT1_1 with a reciprocal increase of NEAT1_2: 78

2.3.3 Nuclear paraspeckles can form in GBM and deletion of the NEAT1 PAS resulted in paraspeckle hyperformation: 81

2.3.4 The nuclear RBP QKI-5 regulates NEAT1 isoforms reciprocally: 85

2.3.5 The QREs located near the NEAT1 PAS mediate the regulation of NEAT1 isoform biogenesis by QKI-5: 89

2.3.6 Reciprocal alterations of NEAT1 isoforms upon ΔPAS induce broad changes of the GBM transcriptome and gene pathways: 93

2.3.7 The increase of NEAT1_2 caused by NEAT1 PAS deletion is responsible for promoting glioma cell migration: 97

2.4 Discussion and conclusions: 100

2.5 Experimental procedures: 107

2.5.1 Cell culture and transfection: 107

2.5.2 Antisense oligonucleotide transfections: 108

2.5.3 Plasmids: 109

2.5.4 RNA isolation: 109

2.5.5 Poly(A) RNA isolation: 110

2.5.6 DNase treatment: 110

2.5.7 Quantitative RT-PCR (RT-qPCR): 110

2.5.8 RNA-seq and analysis: 111

2.5.9 Splicing efficiency analysis: 111

2.5.10 Fluorescence in situ hybridization (FISH): 112

2.5.11 Microscopy and image analysis: 113

2.5.12 Immunoblotting: 113

2.5.13 Transwell migration assay: 114

2.5.14 Statistical analysis: 114

2.5.15 Data availability: 114

2.6 Supplemental information: 115

2.6.1 Special Note: 115

2.7 Acknowledgements: 126

2.8 Author contributions: 126

2.9 Funding: 126

2.10 Conflict of interest: 127

2.11 Abbreviations: 127

CHAPTER 3: THE SCHIZOPHRENIC RISK FACTOR LONG NONCODING RNA GOMAFU IS REGULATED BY THE RNA-BINDING PROTEIN QUAKING

3.1 Abstract: 129

3.2 Introduction: 130

3.3 Results: 131

3.3.1 The nuclear RNA-binding protein QKI regulates GOMAFU formation: 131

3.3.2 Preferential abundance in GOMAFU transcript levels and stability: 135

3.3.3 QKI-5 protein is the predominant isoform and loss of QKI-5 changes QKI-6 and QKI-7 mRNA levels at steady-state: 137

3.3.4 Loss of QKI-5 does not alter the cellular distribution of GOMAFU-interacting RBPs: 139

3.3.5 miR-137 may be a regulator of QKI-5: 141

3.4 Discussion and conclusions: 143

3.5 Materials and methods: 144

3.5.1 Cell culture and transfection: 144

3.5.2 RNA isolation: 145

3.5.3 Quantitative RT-PCR (RT-qPCR): 145

3.5.4 Immunofluorescence: 145

3.5.5 Microscopy and image analysis: 146

3.5.6 Immunoblotting: 146

3.5.7 Prediction of miR-137 interaction sites: 147

3.5.8 Statistical analysis: 147

3.6 Supplemental Information: 148

3.7 Acknowledgements: 148

3.8 Funding: 149

3.9 Conflict of interest: 149

CHAPTER 4: DISCUSSION AND FUTURE DIRECTIONS

4.1 Overview of work discussed: 151

4.2 Nuclear QKI-5 functions to regulate NEAT1 and GOMAFU in different systems: 152

4.3 Proper detection of lncRNA levels is essential for investigating the multifaceted roles and dysregulation of lncRNAs in human diseases: 155

4.4 Dysregulation of NEAT1 isoforms influences paraspeckle formation and the glioma transcriptome: 157

4.5 GOMAFU functions to organize RBPs in a human NPC cell model: 160

4.6 Future Directions: 160

4.7 Final Remarks: 162

REFERENCES: 164

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	Biology, Cell Biology, Molecular Biology, Genetics
Parola chiave	GOMAFU RNA-binding Protein RNA Biology QKI Schizophrenia Glioblastoma NEAT1 Long noncoding RNA
Committee Chair / Thesis Advisor	Yue Feng, Emory University
Committee Members	Homa Ghalei, Emory University Anita H. Corbett, Emory University Graeme L. Conn, Emory University Bing Yao, Emory University

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