The quaking I Pathway and Regulation of Alternative Splicing in Myelinating Glia Open Access

Mandler, Mariana Dalit (2014)

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Splicing, exon ligation and intron removal from pre-mRNA, is fundamental for protein production in eukaryotic cells. Alternative splicing (AS), the differential inclusion of exons to increase proteome diversity, occurs for 95% of expressed mammalian genes. AS is rigorously regulated during normal cell growth and development, particularly in the central nervous system (CNS). Dysregulation of AS is observed in several types of cancer, and is implicated in psychiatric disorders and neurodegenerative diseases (NDDs). Specifically, numerous genes expressed in oligodendroglia (OLs), the myelinating glia of the CNS, undergo extensive regulation by AS. However, the underlying mechanisms are poorly understood. The selective RNA-binding protein quaking I (QKI) is critical for AS in OLs. Three main QKI isoforms display differential nuclear-cytoplasmic distribution, nuclear QKI-5, and cytoplasmic QKI-6 and 7, and play distinct functions. QKI deficiency in OLs results in severe AS defects. However, how QKI controls AS remains elusive. Additionally, mechanism(s) that determine QKI isoform expression are unknown. I found that QKI deficiency results in abnormal upregulation of splicing factors (SFs) in OLs that cause aberrant AS, which are rescued by the cytoplasmic QKI-6. QKI-6 targets the mRNAs of SFs directly or likely involving microRNAs, and inhibits mRNA translation, which contributes to the developmental downregulation of SFs during OL differentiation. Some QKI-target SFs are also implicated in cancer and NDDs. During differentiation of OL progenitor cells, QKI mRNA and protein isoforms are differentially up-regulated, suggesting translation regulation of QKI isoform expression during OL differentiation. In contrast, QKI mRNA isoforms display different profiles in glial and neuronal progenitors, suggesting regulation of QKI mRNA biogenesis and/or stability during neural lineage specification. Finally, I identified a role for the SF FOX2 in controlling QKI-7 mRNA levels in OLs. These studies unveiled novel pathways controlling AS in myelinating glia. I provided the first evidence that QKI-6 is a major regulator of SF expression dictating AS in OLs. Furthermore, I uncovered potential mechanisms that underlie QKI isoform expression during neuron-glia cell fate specification and OL differentiation. Future investigation will define the role of QKI in cancer and brain disorders that harbor QKI deficiency and AS abnormalities.

Table of Contents

Chapter 1: Introduction to Dissertation 1

1.1 The discovery and function of alternative splicing (AS) 4

1.1.1 The splicing process and core machinery in eukaryotic gene expression 4

1.1.2 AS fosters proteome diversity in higher eukaryotes 5

1.1.3 Regulation of AS to control gene expression 13

1.1.4 Dysregulation of AS implicated in disease 18

1.2 AS in the nervous system 20

1.2.1 AS in the development and function of neuronal cell types 20

1.2.2 Functional roles for AS in myelinating glia 23

1.2.3 Dysregulation of AS in CNS/PNS disorders 30

1.3 Roles for splicing factors hnRNP F and H in oligodendroglia (OLs) 32

1.3.1 hnRNP F and H are functional orthologues that regulate RNA metabolism 32

1.3.2 hnRNP F/H can regulate AS in a cell-type specific manner 39

1.3.3 Functions for hnRNP F/H in OL differentiation 41

1.3.4 Dysregulation of hnRNP F/H in disease 42

1.4 Function and importance of selective RNA-binding protein quaking I (QKI) 44

1.4.1 Regulation of QKI isoform expression 44

1.4.2 Functional dependence of the QKI isoforms on sub-cellular localization 48

1.4.3 Alterations of QKI expression in human disease 52

1.5 Summary 56

Chapter 2: A cytoplasmic quaking I isoform regulates the hnRNP F/H-dependent alternative splicing pathway in myelinating glia 60

2.1 Introduction 61

2.2 Results 63

2.2.1 QKI-6 deficiency in OLs is responsible for aberrant over-expression of hnRNP F/H proteins without affecting their mRNAs 63

2.2.2 hnRNP F/H targets G-run elements to regulate inclusion of the alternative exon in the MAG pre-mRNA, which is dysregulated in the qkv/qkv mutant 75

2.2.3 QKI deficiency affects the hnRNP F/H-dependent AS pathway in myelinating glia of the CNS and PNS 87

2.3 Discussion 98

Chapter 3: The regulation of splicing factor (SF) expression by QKI-6 103

3.1 Introduction 104

3.2 Results 107

3.2.1 QKI-6 inhibits translation of hnRNP F/H in myelinating glia 107

3.2.2 QKI deficiency in OLs leads to dysregulation of PTBP1 and PTBP2 levels 118

3.2.3 QKI deficiency affects expression of additional SFs that are implicated in human disease 124

3.3 Discussion 133

Chapter 4: Exploring mechanisms that regulate QKI isoform expression 138

4.1 Introduction 139

4.2 Results 142

4.2.1 mRNA translation underlies QKI isoform expression in OPC differentiation 142

4.2.2 Steady-state levels of QKI mRNAs display differential patterns in mouse neuronal and OL cell lines 145

4.2.3 Knockdown of splicing factor FOX2 specifically reduces QKI-7 mRNA levels 148

4.3 Discussion 156

Chapter 5: Conclusions and Future Directions 160

5.1 A novel AS pathway controlled by QKI-6 in myelinating glia 166

5.2 QKI-6 as a major regulator of splicing factor expression 170

5.3 Potential mechanisms controlling QKI isoform expression 175

5.4 Emerging studies on the mechanisms regulating AS of pre-mRNA 180

5.5 Future directions and implications for human health and disease 183

Chapter 6: Material and Methods 186

6.1 Chapter 2 187

6.2 Chapter 3 192

6.3 Chapter 4 195

Chapter 7: References 199

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