Defining the function of the polyadenosine RNA-binding protein ZC3H14: Molecular insight into a disease associated RNA-binding protein Open Access

Morris, Kevin (Spring 2018)

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One critical step for proper eukaryotic gene expression involves the successful

execution of nuclear RNA processing events that are coupled to efficient export of the

resulting messenger ribonucleoprotein (mRNP). Once properly processed in the nucleus

the resulting mature mRNP is exported to the cytoplasm and is subject to ongoing

regulation as the mRNA is targeted for transport, translation or decay. RNA-binding

proteins are critical for assembling mature mRNPs and facilitating mRNA processing. In

this role, RNA-binding proteins both ensure that mRNAs are properly processed and target

defective RNAs for decay. The critical need for RNA-binding proteins is evidenced by the

growing number of diseases that result from mutations in genes encoding these RNAbinding

proteins. One key RNA-binding protein is the zinc finger polyadenosine RNAbinding

protein, ZC3H14. Loss of function mutations in the ZC3H14 gene causes a nonsyndromic

form of inherited, autosomal recessive intellectual disability. As ZC3H14 is

ubiquitously expressed, this finding suggests that there is a critical role for ZC3H14 in the

brain. However, the molecular function of the protein has yet to be determined. To probe

the function of ZC3H14 in the brain, we carried out a proteomic study to identify the

complete spectrum of ZC3H14 co-purifying proteins. From this approach, we identified

numerous RNA processing factors that allow us to better understand the role ZC3H14

could play in the brain. By combining biochemical and molecular approaches, we assessed

the importance of ZC3H14 and a variety of mRNA factors. We established links to

ZC3H14 and the proper control of splicing, 3’end processing and nuclear export. To that

end, we have provided insight into how ZC3H14 and other interacting proteins influence post-

transcriptional events of gene expression in the brain.

Table of Contents

Table of Contents:

Chapter 1: The critical role of RNA-binding proteins in regulating proper gene


1.1 Posttranscriptional control of gene expression 2

1.1.1 Nuclear mRNA processing events 4 Capping of eukaryotic mRNAs 4 Splicing of eukaryotic pre-mRNA 5 Alternative splicing of eukaryotic RNA transcripts 5 3’ end formation of eukaryotic mRNA 6 Nuclear Export of mRNAs 7

1.1.2 Coordination of nuclear RNA processing events 7 RNA Polymerase II is a source for coupling mRNA processing events 8 Coupling of transcription to splicing and nuclear export 9 Coupling splicing, 3’ end formation, and nuclear export 10 Coupling alternative splicing to 3’end formation 10

1.1.3 Quality control of nuclear processing events 11 SR proteins as mediators of mRNA stability 12 Monitoring of mRNA processing by the RNA exosome and the TREX


12 mRNP surveillance and remodeling at the NPC 13

1.2 Importance of RNA-binding proteins in mRNA processing 14

1.2.1 RNA-binding proteins and human disease 15 The polyadenosine RNA-binding protein ZC3H14 is linked to intellectual



1.2.2 The RNA-binding Protein ZC3H14 is evolutionarily conserved 17 Nab2, the budding yeast orthologue of ZC3H14 is important for mRNA


18 dNab2, the fly orthologue of ZC3H14 has an important role in the brain 18 ZC3H14 protein has a specialized role in regulating mRNA processing in

the brain


1.3 Using protein interaction networks to study human disease 20

1.3.1 The majority rule of determining protein function 20

1.3.2 Identifying protein binding partners using mass-spectrometric analysis 22

1.4 Research Questions to be answered and Innovation 23

Chapter 2: ZC3H14 interacts with a variety of RNA regulatory factors

2.1 Summary of Chapter 27

2.2 Introduction 28

2.3 Materials and Methods 29

2.4 Results 32

2.4.1 ZC3H14 interacts with a variety of mRNA processing factors 34

2.4.2 ZC3H14 interacts with proteins in both an RNA-independent and RNAdependent



2.4.3 Isoform of ZC3H14 has an isoform specific phosphorylation site 37

2.4.4 ZC3H14 forms non-canonical mRNPs with metabolic enzymes. 37

2.5 Discussion 38

Chapter 3: The Polyadenosine RNA-Binding Protein, ZC3H14 Interacts with the THO

Complex and Coordinately Regulates the Processing of Neuronal Transcripts

3.1 Summary 49

3.2 Introduction 51

3.3 Materials and Methods 53

3.4 Results 59

3.4.1 ZC3H14 interacts with the THO complex 59

3.4.2 Loss of ZC3H14 and THO components affect processing of mRNAs 63

3.5 Discussion 70

Chapter 4: Discussion of findings and future perspectives

4.1 Summary of presented studies 91

4.2 Conclusions from our presented studies 95

4.2.1 Linking mutations in ZC3H14 to intellectual disability 96

4.3 Further perspectives on the function of ZC3H14 98

4.3.1 Role of ZC3H14 in nuclear mRNA processing 98 How ZC3H14 and THO components can affect poly(A) tail length 99 ZC3H14 ensures proper processing of target transcripts 99 How ZC3H14 prevents the escape of pre-mRNA to the cytoplasm 100

4.3.2 Role of ZC3H14 in the cytoplasm 101

4.3.3 The role of ZC3H14 in non-canonical mRNPs 103

4.4 Future research in examining the function of ZC3H14 104

4.4.1 Characterizing the mRNA processing defects resulting from loss of ZC3H14


4.4.2 Testing whether ZC and R- loops 105

4.4.3 Examining the requirement for ZC3H14 in local translation 106

4.4.4 Future of the ZC3H14 mutant mouse model 107 Examining the Zc3h14 mutant mouse along with the Thoc1 mutant mouse 107 ZC3H14 and FMRP mouse models 108

4.5 Conclusion and final thoughts. 109

5.0 References 113

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