The quaking I pathway and regulation of alternative splicing in myelinating glia Open Access

Mandler, Mariana Dalit (2014)

Permanent URL: https://etd.library.emory.edu/concern/etds/3x816n37j?locale=en
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Abstract

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

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 diseases.........................................................................................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

List of Figures

Figure 1-1: The spliceosome can be redirected by SFs to cause AS and form multiple mRNAs..........................................................................................................7-8

Figure 1-2: Major types of pre-mRNA processing governed by AS and/or APA.......10-11

Figure 1-3: Structures of three qRRM domains in hnRNP F solved by NMR.............34-35

Figure 1-4: Proposed mode of regulation by hnRNP F/H in regulating AS.................37-38

Figure 1-5: AS of the 3' end of QKI pre-mRNA.........................................................45-46

Figure 1-6: Protein domains of the QKI isoforms........................................................50-51

Figure 1-7: The QKI pathway and regulation of AS in OLs........................................58-59

Figure 2-1: Putative quaking response elements (QRE) are conserved between rodents and human.................................................................................................65-66

Figure 2-2: QKI-6 binds hnRNP H mRNA in a QRE-dependent manner, but not hnRNP F mRNA....................................................................................................67-68

Figure 2-3: Co-immunoprecipitation with QKI-6 by the hnRNP H mRNA under denaturing conditions is attenuated without UV crosslinking..................69-70

Figure 2-4: QKI-6 deficiency results in over-expression of hnRNP F/H in OLs during CNS myelin development without affecting the abundance of hnRNP F/H mRNAs......................................................................................................73-74

Figure 2-5: G-run motifs are identified surrounding MAG Exon 12 in rodents and human........................................................................................................76-77

Figure 2-6: hnRNP F/H promotes inclusion of Exon 12 in MAG pre-mRNA.............78-79

Figure 2-7: hnRNP F/H regulates AS of a MAG minigene in OL and neuronal cell lines...........................................................................................................81-82

Figure 2-8: Inclusion of MAG Exon 12 is reduced upon deletion of intronic G-runs............................................................................................................83-84

Figure 2-9: Deletion of G-runs in the MAG minigene results in comparable reduction of Exon 12 inclusion regardless the knockdown of hnRNP A1....................85-86

Table 2-1............................................................................................................................89

Figure 2-10: Alternative splicing of hnRNP F/H target mRNAs are dysregulated in the qkv/qkv mouse..........................................................................................90-91

Figure 2-11: hnRNP F/H-dependent alternative splicing of MBNL1 and ATXN2 in neuronal and OL cell lines.......................................................................94-95

Figure 2-12: The hnRNP F/H targets are differentially affected in Schwann cells as compared to OLs by QKI deficiency.......................................................96-97

Figure 3-1: Polyribosome association profiles of hnRNP F/H mRNAs in the brain stems of qkv/qkv mutant and the qkv/wt littermate..........................................110-111

Table 3-1: Putative microRNAs targeting the 3'UTR of hnRNP F mRNA in mouse......................................................................................................112-113

Figure 3-2: Abnormal increase in hnRNP F/H detected in qkv/qkv nuclear extracts..................................................................................................114-115

Figure 3-3: hnRNP F/H levels are not elevated in the heart of qkv/qkv mice...........116-117

Figure 3-4: QKI-6 can be UV cross-linking immunoprecipitated (CLIPed) with PTBP1 and PTBP2 mRNAs in brain OLs.........................................................120-121

Figure 3-5: QKI deficiency leads to an abnormal increase in PTBP1 and PTBP2 protein, without affecting steady-state mRNA levels........................................122-123

Figure 3-6: QKI deficiency leads to an abnormal increase in hnRNP M protein levels but not TDP-43............................................................................................127-128

Figure 3-7: Abnormal PSF expression detected in brain stems of qkv/qkv mice......129-130

Figure 3-8: Lack of NLS from PSF results in increased formation of cytoplasmic aggregates in OLs..................................................................................131-132

Figure 4-1: Increase in QKI mRNA levels does not recapitulate protein levels during early OL progenitor cell (OPC) differentiation.....................................143-144

Figure 4-2: Steady-state mRNA levels of QKI isoforms in neuronal and OL cell lines.......................................................................................................146-147

Figure 4-3: FOX2 knockdown reduces QKI-7 mRNA expression..........................150-151

Figure 4-4: FOX2 protein levels are unaffected by QKI deficiency in brain stem of qkv/qkv mutant mice..............................................................................152-153

Figure 4-5: A QKI minigene construct is established, which can be used to determine the regulation of AS of QKI terminal coding exons...................................154-155

Figure 5-1: Diagram of the QKI pathway in neuronal cells.....................................162-163

Figure 5-2: Cytoplasmic QKI-6 controls AS in myelinating glia by suppressing expression of SFs, likely by inhibiting mRNA translation...................164-165

Figure 5-3: Model for APA to determine QKI isoform expression.........................178-179

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