Characterization of disease-relevant targets of RNA binding proteins Pubblico

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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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Biochemistry, Cell & Developmental Biology
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Molecular
Parola chiave	AU-rich element binding proteins Post-transcriptional processing AU-rich elements Poly(A) binding proteins Polyadenosine RNA binding proteins
Committee Chair / Thesis Advisor	Corbett, Anita, Emory University
Committee Members	Powers, Maureen, Emory University Vertino, Paula M, Emory University Bassell, Gary, Emory University Doetsch, Paul, Emory University Ortlund, Eric, Emory University

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