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

Williams, Kathryn Renae (2016)

Permanent URL: https://etd.library.emory.edu/concern/etds/44558d98v?locale=en
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Abstract

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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