Regulation of microRN A-124 biogenesis during human neuronal development Público
Suster, Izabela (Fall 2021)
Published
Abstract
Precise renewal and differentiation of multipotent neural progenitor cells (NPCs), the cells of the central nervous system that differentiate into neurons and certain glial subtypes, must be tightly controlled for normal brain development and function. Abnormalities in NPC renewal and neuron-glia lineage establishment are increasingly being recognized as contributors to the pathogenesis of neuropsychiatric diseases, such as schizophrenia and major depression. A number of neuronal-lineage specific microRNAs (miRNAs/miRs), represented by brain-enriched miR-124, have been shown to promote differentiation of NPCs. Despite the well-studied multifaceted regulation of miR-124 biogenesis in rodent neurons, molecular mechanisms that control miR-124 biogenesis during human neuronal development remain largely undefined. In this dissertation, the transcriptional and posttranscriptional mechanisms regulating the biogenesis of pro-neurogenic miRNA-124 in human NPCs (hNPCs) and neurons are explored.
Human and mouse miR-124 are encoded by three distinct loci that give rise to three primary miR-124 transcripts, pri-miR-124-1, -2 and -3, all of which can be processed into mature miR-124. We examined the expression profile of the human pri-miR-124 paralogs in early neurodevelopment. We show that in contrast to mouse embryonic stem cells, which predominantly express pri-miR-124-1, hNPCs predominantly express pri-miR-124-2. We identified a human-specific cis regulatory element proximal to the miR-124-2 host gene promoter, which undergoes a developmental change in chromatin accessibility and scaffolds transcriptional activators and repressors to regulate transcription of the miR-124-2 host gene during neuronal differentiation.
We next explored posttranscriptional regulation of pri-miR-124-2 at the Microprocessor cleavage step. We discovered that pri-miR-124-2 harbors a binding site for the RNA-binding protein, Quaking (QKI), which is selectively expressed in NPCs and glia. Our data demonstrate that elimination of the nuclear QKI isoform in a hNPC cell line model increases levels of mature miR-124. Furthermore, we detect rapid downregulation of QKI upon hNPC differentiation into neurons, which should lead to increased miR-124 production. Together, these studies define novel mechanisms that underlie miR-124 biogenesis to advance early human neuronal development.
Table of Contents
Abbreviations
Chapter 1: Introduction to Dissertation 1
Introduction 2
1.1 Canonical and non-canonical miRNA processing pathways 3
1.1.1 The canonical miRNA biogenesis pathway 3
1.1.2 Emerging roles of non-canonical miRNA biogenesis
pathways in the central nervous system 5
1.2 MiRNAs play key roles in governing neuronal development 6
1.2.1 The most abundant miRNA in the brain: miR-124 and its
anti-neurogenic targets 6
1.2.2 Convergence of distinct pro-neurogenic miRNAs on key inhibitors of neuronal differentiation 8
1.3 Neural miRNA multigene families 11
1.4 Regulation of miRNAs in neural development: divergence by biogenesis
and convergence on key targets 13
1.4.1 Transcriptional regulation of miRNA genes 13
1.4.2 Posttranscriptional regulation of miRNA biogenesis 16
1.4.3 Posttranscriptional regulation of miRNA activity 18
1.5 Emerging evidence for miRNA dysregulation in brain disorders:
dysregulation, etiology and therapeutic potential 20
1.6 Questions Addressed in this Work 23
Figures
Figure 1-1: The miR-124-PTBP1 regulatory loop 26
Figure 1-2: The human microRNA-124 multigene family 28
Figure 1-3: Transcriptional regulation of microRNA host genes 30
Figure 1-4: Posttranscriptional regulation of pri-miRNAs 32
Chapter 2: Transcriptional regulation of a hNPC-specific pri-miR-124 paralog in early human neuronal lineage development 34
2.1 Abstract 35
2.2 Introduction 36
2.3 Results 39
2.3.1 Pri-miR-124-2 is the primary source for miR-124
biogenesis in hNPCs 39
2.3.2 Unlike pri-miR-124-1, pri-miR-124-2 processing is not
inhibited by PTBP1 40
2.3.3 Identification of a human-specific proximal promoter
element that regulates MIR-124-2HG transcription during differentiation of hNPCs 41
2.3.4 Functional coordination of a transcriptional activator and
repressor at the MIR124-2HG locus during neuronal maturation 43
2.4 Discussion 45
Figures and Tables Figure 2-1: MIR-124-2 is the predominant source of miR-124
in hNPCs. 49
Figure 2.2: PTBP1 does not inhibit processing of pri-miR-124-2. 51
Figure 2-3: Chromatin accessibility of the MIR124-2HG proximal
promoter element increases during neuronal differentiation. 53
Figure 2-4: MAFK negatively regulates transcription through the
MIR124-2HG proximal promoter element. 55
Figure 2-5: SP1 controls miR-124 biogenesis in hNPCs. 57
Figure 2-6: Model of developmentally regulated chromatin and
transcription factor dynamics at the MIR124-2HG locus in human
NPCs and neurons. 59
Supplementary Figure S2.1: A switch between pri-miR-124 paralogs
in the developing mouse cerebellum, recapitulates human
cerebellar development. 61
Supplementary Figure S2.2: The human MIR124-2HG promoter
region and developmentally programed chromatin accessibility. 63
Supplementary Figure S2.3: Differential expression of candidate
TFs during in vitro neuronal differentiation 65
Supplementary Figure S2.4: SP1 decreases in BE(2)-M17 cells
having undergone 0 (D0) and 10 days (D10) of retinoic acid-
induced differentiation. 67
Supplemental Table S2.1: Human FANTOM5 sample descriptions and data values 69
Supplemental Table S2.2: Mouse FANTOM5 sample descriptions and data values 70
Supplemental Table S2.3: Oligo sequences used in this study. 72
Chapter 3: Posttranscriptional mechanisms regulating miR-124 biogenesis during neurodevelopment 73
3.1 Introduction 74
3.2 Results
3.2.1 Expression of miRNA machinery during in vitro differentiation
of BE(2)-M17 cells 77
3.2.2 In silico prediction program identifies QKI as a candidate regulator of pri-miR-124-2 processing 78
3.2.3 QKI is expressed in hNPCs, declining before PTBP1 during neuronal differentiation 80
3.2.4 QKI does not associate with DGCR8 of the Microprocessor complex in neural cells 81
3.2.5 Deletion of nuclear QKI5 increases mature miR-124 levels 82
3.3 Discussion 82
Figures and Tables
Figure 3-1: Expression of Microprocessor subunits is unchanged
during in vitro differentiation of BE(2)-M17 cells 84
Figure 3-2: Human pri-miR-124-2 harbors a QRE 86
Figure 3-3: QKI is expressed in hNPCs and declines before PTBP1
during neuronal differentiation. 88
Figure 3-4: QKI does not interact with DGCR8 in BE(2)-M17 neuroblastoma cells. 90
Figure 3-5: Deletion of nuclear QKI5 increases expression of mature
miR-124. 92
Figure 3-6: A schematic representation of how nuclear QKI5 may
regulate processing of pri-miR-124-2 by the Microprocessor
complex in hNPCs. 94
Supplementary Table S3.1: List of CRIP predicted RBPs with
binding sites in the pri-miR-124-2 region. 96
Table S3.2 – Oligo sequences used in this study. 98
Chapter 4: Conclusions and Future Directions 99
4.1 Potential mechanisms regulating the preferential expression of
pri-miR-124-2 in human neural progenitor cells 100
4.2 How does neuronal differentiation persist in DmiR-124 iPSC-derived neurons? 104
4.3 Deciphering the role of individual pri-miR-124 paralogs in advancing neuronal differentiation 106
4.4 Potential mechanisms downregulating miR-124-2 during murine
neuronal differentiation 108
4.5 Potential mechanisms mediating regulation of human pri-miR-124-2 processing by QKI5 110
Chapter 5: Materials and Methods 112
Chapter 6: References 123
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