Characterization of disease-relevant targets of RNA binding proteins Öffentlichkeit
Wigington, Callie (2015)
Abstract
Messenger RNA (mRNA) serves as the intermediate molecule in the flow of genetic information from DNA to protein and is subject to extensive regulatory events, collectively referred to as post-transcriptional processing. These events include the capping, splicing, 3' end processing, export and eventual decay of an mRNA transcript. Each of these processing events is mediated by a host of post-transcriptional factors, including RNA binding proteins and noncoding RNAs. The critical nature of these steps in ensuring proper gene expression is supported by the observation that dysregulation of many RNA binding proteins is associated with a variety of human diseases, including intellectual disability and cancer. Therefore, the functional characterization of newly discovered RNA binding proteins as well as the identification of novel targets for canonical RNA binding proteins is an important objective to understand the underlying molecular pathogenesis of disease. In this work, we demonstrate that the novel polyadenosine RNA binding protein, ZC3H14, specifically interacts with and modulates the pre-mRNA processing of the ATP5G1 transcript, which encodes a key ATP synthase subunit. Consistent with a loss of ATP synthase activity, we observe reduced cellular ATP levels and a striking mitochondrial fragmentation phenotype upon knockdown of ZC3H14. We hypothesize that these defects in cellular ATP levels and mitochondrial morphology may, at least in part, underlie the observed intellectual disability in patients with loss-of-function mutations in ZC3H14. In this work, we also present a novel mode of post-transcriptional regulation of the PDCD4 transcript, which encodes a novel tumor suppressor that is downregulated in a number of cancer types, including breast. We demonstrate that the two well-characterized U-rich RNA binding proteins, HuR and TIA1, compete for interaction with overlapping binding sites on the PDCD4 3'UTR, resulting in positive regulation of PDCD4 mRNA and protein levels in breast cancer cells. The findings presented here not only demonstrate the importance of post-transcriptional processing events in ensuring precise gene expression, but also provide evidence that the two transcripts analyzed in these studies play important roles in the molecular pathogenesis of disease.
Table of Contents
Chapter 1: Introduction 1
Section 1
Regulation of gene expression by post-transcriptional processing 2
Factors that mediate post-transcriptional processing events 8
RNA binding proteins in human disease 10
Section 2
The poly(A) tail: discovery and addition 12
Canonical poly(A) binding proteins 14
ZC3H14 is a novel poly(A) RNA binding protein 20
ZC3H14: Insight from model organisms 22
ZC3H14 is important for proper neuronal function 24
Defining a role for ZC3H14 in mRNA processing Section 3 25
The 3' untranslated region 28
AU-rich elements and the factors that bind them 31
HuR and TIA1, two U-rich element binding proteins 33
HuR and TIA1 binding studies: targets and recognition 38
HuR and TIA1 as coordinate regulators of gene expression 41
HuR and TIA1 in cancer 42
Scope and significance of this dissertation 44
Tables and Figures 47
Chapter 2: ZC3H14 Modulates the pre-mRNA Processing of ATP5G1 mRNA 56
Introduction 57
Results 60
Discussion 74
Tables and Figures 80
Experimental Procedures 92
Chapter 3: Investigating Multiple Aspects of ZC3H14 Regulation 99
Introduction 100
Results 102
Discussion 113
Tables and Figures 117
Experimental Procedures 125
Chapter 4: Post-transcriptional Regulation of PDCD4 mRNA by HuR and TIA1 130
Introduction 131
Results 135
Discussion 154
Tables and Figures 163
Experimental Procedures 179
Chapter 5: Discussion 192
Brief overview 193
ZC3H14: Implications for mRNA processing and the brain 194
Integrating the roles of multiple Pabs in human cells 200
Implications for HuR and TIA1 in breast cancer: PDCD4 is a relevant target 202
Future directions 205
Final conclusions 207
Figures 209
References 211
About this Dissertation
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Primary PDF
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Characterization of disease-relevant targets of RNA binding proteins () | 2018-08-28 14:33:20 -0400 |
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Supplemental Files
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Chapter 5 Figures.pdf () | 2018-08-28 14:36:29 -0400 |
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Chapter 2 Figures.pdf () | 2018-08-28 14:37:55 -0400 |
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Chapter 4 Figures.pdf () | 2018-08-28 14:39:27 -0400 |
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Chapter 1 Figures.pdf () | 2018-08-28 14:40:54 -0400 |
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Chapter 3 Figures.pdf () | 2018-08-28 14:42:41 -0400 |
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