Regulation of microRN A-124 biogenesis during human neuronal development Open Access

Suster, Izabela (Fall 2021)

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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


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


           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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