The Drosophila Nab2 RNA-Binding Protein Regulates Splicing Neuronal RNAs Open Access
Jalloh, Binta (Spring 2021)
Abstract
A large number of RNA binding proteins (RBPs) play important roles in RNA processing and in regulating gene expression at the post-transcriptional level. It is therefore not surprising that dysregulation of key RNA processing steps due to mutations in genes that encode these RBPs are often linked to tissue-specific diseases. A majority of which affects the highly specialized neuronal cell, resulting in neurodevelopmental and or neurodegenerative disorders. One such RBP that is essential for neurons and required for proper brain development and function is the polyadenosine RNA-binding protein, ZC3H14 (Zinc finger Cysteine-Cysteine-Cysteine-Histidine containing protein 14). Mutations in the gene encoding ZC3H14 has been linked to a heritable and a non-syndromic, autosomal recessive form of intellectual disability. To investigate the role(s) of ZC3H14 in the brain, we employed the Drosophila melanogaster ortholog of ZC3H14, Nab2, to study which RNAs are affected upon Nab2 loss. We show here that Drosophila Nab2 is implicated in splicing. Using RNA from adult Drosophila heads, we performed the first global transcriptomic analysis on Nab2 heads using RNA sequencing experiments. The Nab2 mutants show defects in RNA splicing compared to controls. Further analysis comparing mutant females to controls reveal dysregulation of several pathways that are important for proper brain function and sheds light into which RNA transcripts are most affected by the loss of Nab2 in neuron enriched tissues. This high-throughput and unbiased RNA sequencing dataset provides a novel functional role for Drosophila Nab2 in brain tissue by regulating the splicing of a specific subset of RNAs. Of the ~150 affected neuronal RNAs, the most significant splicing defects observed is the female-specific retention of a male-specific exon in the sex determination factor gene, Sex lethal.
Table of Contents
Chapter 1: General Introduction.....................................................................................1
1.1 The Central Dogma of Molecular Biology....................................................................1
1.2 Regulation of Gene Expression at the RNA Level .......................................................2
1.3 Gene Expression Is Modulated By Post-Transcriptional Regulation……................…3
1.4 Post-Transcriptional Regulation By RNA-Binding Proteins (RBPs)…………….…...6
1.5 Zinc Finger (ZnF) Poly(A) Binding Proteins (PABPs)…………………………..…...7
1.6 PABPs In Human Diseases………………………………………………………...….9
1.7 ZC3H14 Is An Evolutionarily Conserved PAB Proteins…………………......……...10
1.7.1 Structure and Function of ZC3H14/Nab2 Protein………………...….........11
1.7.2 ZC3H14 Controls Poly(A) Tail Length………………………….………...12
1.8 A Role for ZC3H14 In RNA Splicing………………………………………….........13
1.9 Drosophila Melanogaster as A Model to Study Neurological Diseases………….…14
1.9.1 A Drosophila Disease Model to Study ZC3H14 Linked Disease.…….......15
1.10 Role & Requirement For Drosophila Nab2 In Neurons…………………..…….…17
1.11 ZC3H14/Nab2 Show Specificity For Target RNAs…………………………….....19
1.12 Roles & Requirement of m6A RNA Methylation In Neurons………………….....20
1.13 The m6A RNA Methylation Machinery Is A Dynamic Complex…….…………..20
1.14 Summary & Scope………………………………….……………………….........22
1.15 Figures…………………………………………………………………………....25
Chapter 2: The Nab2 RNA-binding protein promotes sex-specific splicing of Sex lethal in Drosophila neuronal tissue………………………………………………..….39
2.1 Abstract........................................................................................................................40
2.2 Introduction..................................................................................................................41
2.3 Results..........................................................................................................................44
2.3.1 Nab2 loss affects levels and processing of a subset of RNAs in the head transcriptome..........................................................................................................44
2.3.2 Nab2 loss alters levels of transcripts linked to mRNA processing……….45
2.3.3 Nab2ex3 females exhibit masculinized Sxl splicing in neuron-enriched tissues……………………………………………………………………………47
2.3.4 The dosage compensation complex contributes to phenotypes in Nab2ex3 mutant female…………………………………………………………………49
2.3.5 Nab2-regulated splicing of Sxl exon3 is dependent upon the Mettl3 m6A methyltransferase…………………………………….……………………….50
2.3.6 Nab2 binds Sxl pre-mRNA and modulates its m6A methylation…………………………………………………………..………52
2.4 Discussion..............................................................................................................54
2.5 Figures...................................................................................................................57
Chapter 3: A Drosophila model of Pontocerebellar Hypoplasia reveals a critical role for the RNA exosome in neurons…………………………………………………………………………………..88
3.1 Abstract..................................................................................................................89
3.2 Author Summary....................................................................................................91
3.3 Introduction............................................................................................................92
3.4 Results....................................................................................................................95
3.4.1 The RNA exosome subunit Rrp40 is essential for development………….95
3.4.2 Rrp40 is required for proper development of the Drosophila mushroom body neurons……………………………………………………………..……..95
3.4.3 Rrp40 is required for age-dependent function in neurons…………………………….…………………………………………....96
3.4.4 PCH1b mutations engineered into the Rrp40 locus impair viability and shorten lifespan………………………………………………………………….97
3.4.5 Rrp40 mutant flies exhibit age-dependent morphological and behavioral defects…………………………………………………………………………..99
3.4.6 Rrp40G11A mutant flies have a mushroom body β-lobe midline-crossing defect……………………………………………………………………………99
3.4.7 Pan-neuronal expression of human EXOSC3 is sufficient to rescue behavioral phenotypes in Rrp40 mutant flies………………………………..…99
3.4.8 Steady-state levels of RNA exosome subunit levels are affected in Rrp40 mutant flies………………………………………………………………….…100
3.4.9 RNA-sequencing analysis of Rrp40 alleles reveals distinct gene expression profiles……………………………………………………………………..…..101
3.4.10 RNA-sequencing analysis reveals increased steady-state levels of key neuronal genes in Rrp40 mutant flies………………………………………….102
3.5 Discussion.................................................................................................................104
3.6 Figures……………………………………………………………………...………112
Chapter 4: Discussion……………………………………………………………….145
4.1 New Insights Into The Central Dogma of Molecular Biology…………...1454.2 Brief Overview of Main Findings………………………………………...146
4.3 Implications of ZC3H14/Nab2 In Regulating mRNAs In Brain Neurons………..1474.4 Exploring A Role For Drosophila Nab2 In Regulating mRNA Splicing………...148
4.4.1 Impact of Nab2 Loss In A Subset of RNAs…………………………....149
4.5 Implication of Nab2 In Regulating mRNAs Via Modulation of m6A RNA Methylation……………………………………………………………………..……149
4.6 Exploring roles of Nab2 in controlling dosage compensation genes in neurons………………………………………………………………………….……151
4.7 The RNA Exosome Regulates Neuronal RNAs………………………………………………………………………….……..152
4.8 Future Direction and Questions………………………...…………………………………………………153
4.9 Final Conclusions………………………………………………………………154
4.10 Summary Model Figure………………………………..……………………………………….….....156
Chapter 5: Material and Methods........................................................................158
5.1 Chapter 2......................................................................................................159
5.2 Chapter 3......................................................................................................167
Chapter 6: References............................................................................................171
List of Figures
Chapter 1:
Figure 1—1: Post-transcriptional regulation of gene expression at the RNA level……………………………………………………………………………………....25
Figure 1—2: RNA-binding proteins and their functions in RNA biology………………………………………………………………………….……..…27
Figure 1—3: Domain structure of the ZC3H14/Nab2 protein family……………………………………………………………………………...…..…29
Figure 1—4: RNA-binding proteins (RBPs) including ZC3H14 and human diseases…………………………………………………………………………..……....31
Figure 1—5: Pedigrees of families with ZC3H14 mutations linked to disease……………………………………………………………………………….…...33
Figure 1—6: Drosophila Nab2 is required for development of mushroom body neurons……………………………………………………………………...………..…..35
Figure 1—7: Dynamic role of m6A in RNA processing paths………………………………………………………………………..…………….37
Chapter 2:
Figure 2—1: RNA sequencing detects effects of Nab2 loss on the head transcriptome……………………………………………………………………………..58
Figure 2—2: Significantly up/down-regulated RNAs in Nab2ex3 heads are enriched for predicted factors……………………………………………………..........................…...60
Table 2—1: Alternative exon usage (DEXSeq) in Nab2ex3 head transcriptomes…….………………………………………………………………..….....62
Figure 2—3: Alternative splicing of Sxl is disrupted in Nab2ex3 female heads………………………………………………………………………………….…63
Figure 2—4: An allele of the DCC component male-specific lethal-2 (msl-2) rescues Nab2 phenotypes in females…………………………………………….…………..….65
Figure 2—5: Removing the Mettl3 m6A transferase suppresses viability, behavioral, neuroanatomical and Sxl splicing defects in Nab2 mutant females…………………...…67
Figure 2—6: Nab2 associates with the Sxl mRNA and inhibits its m6A methylation.…..69
Figure 2—S1: RNA sequencing reads across the Nab2 locus…………………….….….71
Figure 2—S2: GO term enrichment among Nab2-regulated alternative splicing events..73
Figure 2—S3: RNA sequencing reads across the CG13124 and Ih channel loci…….....75
Figure 2—S4: RNA sequencing reads across the tra and dsx loci……….……….…….77
Figure 2—S5: Modification of Nab2ex3 locomotor defect by roX1 and mle alleles.…...79
Figure 2—S6: Genomic PCR confirms the Nab2ex3,Mettl3null recombinant………..….81
Figure 2—S7: Schematic of Sex lethal (Sxl) primers for qPCR…………………….….83
Figure 2—S8: Nab2 limits m6A methylation of additional Mettl3 target RNAs……….85
Tables
Table 2---Supplemental Table 1: DESeq2 results for all genes………………..……..87
Table 2---Supplemental Table 2: MISO called AS events in Nab2 mutant heads………………………………………………………………………………….87
Table 2---Supplemental Table 3: DEXSeq called differential exon usage for Nab2 mutant heads…………………………………………………………………………………87
Chapter 3:
Figure 3—1: The Rrp40 subunit of the RNA exosome is required for viability and proper mushroom body development in Drosophila…………………………………..………..112
Figure 3—2: Rrp40 is required for age-dependent function in neurons……….……….114
Figure 3—3: Generation of Drosophila models of PCH1b amino acid substitutions……………………………………………………………………….…....116
Figure 3—4: Flies that model PCH1b amino acid substitutions in Drosophila show a range of morphological and behavioral phenotypes………………………………………...…118
Figure 3—5: Rrp40G11A mutant flies show defects in mushroom body morphology…...120
Figure 3—6: Locomotor defects in Rrp40 mutant flies are rescued by neuronal expression of human EXOSC3………………………………………………………………..……122
Figure 3—7: Amino acid substitutions that model PCH1b in Rrp40 alter levels of RNA exosome subunits…………………………………………………………………...…..124
Figure 3—8: RNA-seq analysis of Rrp40 mutant flies identifies key neuronal targets that are altered……………………………………………………………………….…..…..126
Figure 3—9: Key neuronal transcripts show an increase in steady-state levels in Rrp40 mutant flies…………………………………………………………………..……..…..128
Figure 3—10: Mechanistic model for how amino acid substitutions could alter RNA exosome function…………………………………………………………………….....130
Figure 3—S1: Validation of Rrp40 RNAi…………………………………..…………..132
Figure 3—S2: CRISPR/Cas9-induced Homology-directed recombination (HDR) with a double-stranded DNA donor vector………………………………….……..…………..134
Figure 3—S3: EXOSC3-PCH1b mutations modeled in Drosophila Rrp40……….….…136
Figure 3—S4: Rrp40G11A and Rrp40D87A mutant flies show overlapping or distinct sets of RNAs…………………………………………………………………………………...138
Figure 3—S5: Integrative Genomic View (IGV) screenshots of multiple functionally important neuronal to illustrate the RNA-Seq data obtained…………..….…………….140
Table 3—S1: Oligonucleotide primer sequence s employed for RT-qPCR……..……..142
S2 Table: RNA-Seq data analysis of Rrp40G11A alleles……………………….……..…144
S3 Table: RNA-Seq data analysis of Rrp40D87A alleles……………………………...…144S1 Video: Negative geotaxis assay for Rrp40wt at Day 4………………………………144
S2 Video: Negative geotaxis assay for Rrp40G11A at Day 4…………………………….144
S2 Video: Negative geotaxis assay for Rrp40D87A at Day 4…………………………….144
Chapter 4:
Figure 4—1: Model summary figure for roles of Drosophila Nab2……………………156
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