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

Williams, Kathryn Renae (2016)

Permanent URL:


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

About this Dissertation

Rights statement
  • Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.
Subfield / Discipline
  • English
Research field
Committee Chair / Thesis Advisor
Committee Members
Last modified

Primary PDF

Supplemental Files