Neuronal Function of hnRNP-Q1: Identification of a Novel Mechanism for Gap-43 mRNA Translation Regulation Open Access

Williams, Kathryn Renae (2016)

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Post-transcriptional regulation of gene expression by mRNA binding proteins is critical for neuronal development and function. hnRNP-Q1 is an mRNA binding protein that was identified as a splicing factor but recent findings demonstrate that hnRNP-Q1 performs critical post-transcriptional regulatory mechanisms in the cytoplasm as well. hnRNP-Q1 has been implicated in mRNA localization, translation and decay modulation. Given that hnRNPQ1 is highly expressed in brain tissue, we hypothesized that hnRNP-Q1 post-transcriptionally represses the expression of specific mRNAs as a means to alter neuron morphology and consequently, function. Here we have identified Growth associated protein 43 (Gap-43) mRNA as a novel target of hnRNP-Q1 and demonstrate that hnRNP-Q1 inhibits Gap-43 mRNA translation and consequently GAP-43 function. GAP-43 is an important neuronal protein that regulates actin dynamics in growth cones and facilitates axonal growth. Previous studies have identified factors that regulate Gap-43 mRNA stability and localization, but it remains unclear whether Gap-43 mRNA translation is also regulated. Our results reveal that hnRNP-Q1 knockdown increased nascent axon length, total neurite length and neurite number in M. musculus embryonic cortical neurons and enhanced Neuro2a cell process extension; phenotypes that were rescued by GAP-43 knockdown. Additionally, we have identified a G-Quadruplex structure in the 5'-UTR of Gap-43 mRNA that directly interacts with hnRNP-Q1 as a means to inhibit Gap-43 mRNA translation. These findings reveal a novel mechanism for regulating GAP-43 expression and function, demonstrate that hnRNPQ1 is a novel G-Quadruplex binding protein and suggest a potential conserved mechanism for hnRNP-Q1-mediated translation inhibition. hnRNP-Q1-mediated inhibition of Gap-43 mRNA translation and potentially additional mRNAs by a similar mechanism may be critical for proper neuronal development, function and regeneration.

Table of Contents

Chapter 1: General Introduction. 1

1.1: mRNA Binding Proteins. 2

1.1.1: mRNA Binding Proteins Regulate mRNA Processing and Post-Transcriptional Regulation. 2

1.1.2: mRNA Binding Protein Dysregulation and Disease. 11

1.2: The hnRNP Family of Proteins. 20

1.2.1: Identification of hnRNP Proteins. 20

1.2.2: Diverse hnRNP Protein Structure. 21

1.2.3: Diverse hnRNP Protein Function. 22

1.3: hnRNP-Q1. 29

1.3.1: hnRNP-Q1 Nuclear mRNA Processing and Post-Transcriptional Regulatory Functions. 31

1.3.2: hnRNP-Q1 Cytoplasmic mRNA Processing and Post- Transcriptional Regulatory Functions. 32

1.3.3: Additional Functions and Regulation of hnRNP-Q1. 35

1.4: Dissertation Hypothesis and Objectives. 36

1.5: Materials and Methods. 37

1.6: Figures. 40

1.7: Supplemental Figures. 47

1.8: Tables. 48

Chapter 2: Identification of Gap-43 mRNA as a Novel hnRNP-Q1 Target. 52

2.1: Introduction. 53

2.1.1: Molecular and Systemic Functions of GAP-43. 53

2.1.2: GAP-43 Expression Regulation.55

2.1.3: Chapter 2 Hypothesis and Objectives. 56

2.2: Results. 57

2.2.1: Elevated GAP-43 Expression in hnRNP-Q1 Deficient N2a Cells. 57

2.2.2: Characterization of Incipient Cortical Neurons. 59

2.2.3: Elevated GAP-43 Expression in hnRNP-Q1 Deficient Primary Cortical Neurons. 60

2.2.4: Inverse Correlation between the Expression of hnRNP-Q1 and GAP-43. 61

2.3: Discussion. 62

2.4: Materials and Methods. 64

2.5: Figures.71

2.6: Supplemental Figures. 80

Chapter 3: hnRNP-Q1 Regulation of GAP-43 Expression Affects Neuron Morphology. 82

3.1: Introduction. 83

3.1.1: Cellular Functions of GAP-43. 83

3.1.2: Cellular Functions of hnRNP-Q1. 85

3.1.3: Chapter 3 Hypothesis and Objectives. 85

3.2: Results. 86

3.2.1: Elevated GAP-43 Expression in hnRNP-Q1 Deficient Cortical Neurons Increased Neurite Length and Number. 86

3.2.2: Increased Focal Adhesions in hnRNP-Q1 Deficient Cortical Neurons. 90

3.2.3: Elevated GAP-43 Expression in hnRNP-Q1 Deficient N2a Cells Increased Process Extension. 91

3.3: Discussion. 92

3.4: Materials and Methods. 95

3.5: Figures. 97

3.6: Supplemental Figures. 105

Chapter 4: Mechanism of hnRNP-Q1-Mediated Regulation of GAP-43 Expression. 116

4.1: Introduction. 117

4.1.1: Potential Gap-43 mRNA Cis-Regulatory Elements. 117

4.1.2: G-Quadruplexes and Translation Regulation. 118

4.1.3: Mechanisms of mRNA Binding Protein Translation Regulation. 119

4.1.4: Chapter 4 Hypothesis and Objectives. 120

4.2: Results. 121

4.2.1: hnRNP-Q1 Directly Binds a G-Quadruplex Sequence in the 5'-UTR of Gap-43 mRNA. 121

4.2.2: hnRNP-Q1 Directly Binds PolyA Stretches and a Consensus Sequence in the 3'-UTR of Gap-43 mRNA. 123

4.2.3: hnRNP-Q1 Binds the Gap-43 5'-UTR G-Quadruplex Sequence through the RGG Box. 124

4.2.4: The Gap-43 5'-UTR G-Quadruplex Sequence Folds into a G-Quadruplex Structure. 125

4.2.5: hnRNP-Q1 Co-localizes with Gap-43 mRNA in Incipient Cortical Neurons. 128

4.2.6: hnRNP-Q1 Represses Endogenous Gap-43 mRNA Translation. 129

4.2.7: hnRNP-Q1 Represses Gap-43 mRNA Translation Through the 5'-UTR G-Quadruplex. 131

4.2.8: A Potential Role for Phosphorylation and miRNA in hnRNP-Q1-mediated Gap-43 mRNA Translation Inhibition. 132

4.3: Discussion. 134

4.4: Materials and Methods. 137

4.5: Figures. 148

4.6: Supplemental Figures. 160

4.7: Tables. 172

Chapter 5: Summary and Future Directions. 173

5.1: Summary. 174

5.2: Future Directions. 175

5.2.1: hnRNP-Q1 Interacts with Multiple Cis-regulatory Elements. 175

5.2.2: Potential Mechanism of hnRNP-Q1-mediated Translation Inhibition. 178

5.2.3: Coordinated Regulation by hnRNP-Q1 and Additional mRNA Binding Proteins. 180

5.2.4: Systemic Functions on hnRNP-Q1-Mediated Post- Transcriptional Regulation. 182

5.3: Concluding Remarks. 183

5.4: Figures. 184

References. 185

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