Tissue-specific regulation of the Pabpn1 gene: Functional implications for muscular dystrophy Open Access
Phillips, Brittany (Spring 2018)
Published
Abstract
Fine-tuned spatio-temporal control of gene expression is often achieved through post-transcriptional regulatory mechanisms via a suite of RNA binding proteins. These RNA binding proteins are responsible for every aspect of RNA processing from 5’ capping, splicing, and 3’ end formation to eventual transcript degradation. Because many of these steps are necessary for proper RNA processing regardless of cell type, many genes encoding these RNA binding proteins are ubiquitously expressed. However, mutations in these genes encoding RNA binding proteins often cause tissue-specific diseases. Defining specific functions of RNA binding proteins in particular tissues will give insight into how mutations in these genes encoding RNA binding proteins cause tissue-specific disease. The nuclear poly(A) binding protein PABPN1, for example, plays critical roles in several RNA processing events including 3’ end formation and polyadenylation. Although the PABPN1 gene is ubiquitously expressed, a small expansion that leads to an increase in an alanine tract in the N-terminus of the PABPN1 protein causes the muscle-specific disease oculopharyngeal muscular dystrophy (OPMD). The work presented in this dissertation explores why specific skeletal muscles are affected by PABPN1 mutations by characterizing novel mouse models of OPMD. These mouse models are critical tools allowing us to dissect the molecular phenotypes underlying OPMD pathology, including assessing the function of expanded PABPN1. Through this work we have determined that the alanine-expanded PABPN1 is only partially functional. As PABPN1 protein levels are very low in muscle compared to non-muscle tissues, a reduction in PABPN1 function could underlie pathology. We have identified multiple factors including the RNA binding protein HuR and microRNAs that regulate Pabpn1 expression in muscle. Our work investigating the post-transcriptional mechanisms that contribute to low PABPN1 levels specifically in muscle provides insight into how PABPN1 expression is regulated and lays the groundwork for novel OPMD therapies seeking to raise PABPN1 levels in muscle. Together, our findings have elucidated how loss of PABPN1 function contributes to OPMD pathogenesis, and how muscle-specific regulation of PABPN1 levels could predispose this tissue to pathology.
Table of Contents
Chapter 1
Introduction
1
1.1
Scope and significance of this dissertation
2
1.2
Co-transcriptional and post-transcriptional regulation of gene expression
5
1.2.1
1 RNA processing: An overview
5
1.2.2
The CTD of the largest subunit of RNA polymerase II mediates co-transcriptional processing
6
1.2.3
The 3’UTR contains cis-regulatory elements that interact with trans-acting factors
8
1.2.4
RNA binding proteins and target recognition
11
1.3
PABPN1: A ubiquitous RNA binding protein that causes tissue-specific disease
13
1.3.1
OPMD: A rare, late-onset muscle disease
13
1.3.2
PABPN1 functions: An overview
14
1.3.3
PABPN1 and 3’ end formation
16
1.3.4
Additional roles for PABPN1 in RNA processing
17
1.4
PABPN1 and OPMD: Understanding tissue-specific disease pathology
19
1.4.1
Aggregates and OPMD
20
1.4.2
Exploring loss of function models of OPMD pathology
22
1.4.3
Muscle-specific Pabpn1 regulation: Implications for OPMD therapies
23
1.5
Figures
25
Chapter 2
Novel mouse models of oculopharyngeal muscular dystrophy (OPMD) reveal early onset mitochondrial defects and suggest loss of PABPN1 may contribute to pathology
31
2.1
Summary
32
2.2
Introduction
33
2.3
Results
36
2.3.1
Generation of a new mouse model of OPMD
36
2.3.2
Pabpn1+/A17 knock-in mice have smaller muscles than wild type littermates
39
2.3.3
Pabpn1+/A17 mice have subtle early-onset RNA phenotypes
40
2.3.4
Proteomic analysis reveals mitochondrial defects in Pabpn1+/A17 mice
42
2.3.5
Comparison with Pabpn1 knock-out suggests loss of function may contribute to but does not fully explain pathology
45
2.4
Discussion
49
2.5
Materials and methods
56
2.6
Figures
66
Chapter 3
Post-transcriptional regulation of Pabpn1 by the RNA binding protein HuR
81
3.1
Summary
82
3.2
Introduction
82
3.3
Results
87
3.3.1
Steady-state Pabpn1 mRNA and protein levels are low in an in vitro model of skeletal muscle
87
3.3.2
The Pabpn1 transcript is unstable in an in vitro model of skeletal muscle
88
3.3.3
3 Pabpn1 alternative polyadenylation does not correlate with stability changes
89
3.3.4
The AU-rich element binding protein HuR interacts with the Pabpn1 3’UTR in vitro
91
3.3.5
HuR interacts with a specific region within the Pabpn1 3’UTR
94
3.3.6
HuR negatively regulates Pabpn1 at the RNA and protein levels
95
3.3.7
HuR negatively regulates Pabpn1 in vivo
96
3.3.8
HuR is more cytoplasmic in C2C12 myotubes than C2C12 myoblasts
97
3.4
Discussion
98
3.5
Materials and methods
103
3.6
Figures
112
Chapter 4
microRNAs regulate Pabpn1 expression in muscle cells
128
4.1
Summary
129
4.2
Introduction
130
4.3
Results
133
4.3.1
The Pabpn1 3’UTR contains multiple putative microRNA binding sites that are conserved
133
4.3.2
microRNA-141-5p negatively regulates Pabpn1 expression
134
4.3.3
microRNA-532-3p positively regulates Pabpn1 expression
135
4.3.4
microRNA-331-3P negatively regulates Pabpn1 expression
136
4.4
Discussion
137
4.5
Materials and methods
140
4.6
Figures and tables
143
Chapter 5
Discussion
152
5.1
Overview
153
5.2
OPMD studies: The limitations of overexpression models
154
5.3
Understanding how loss versus gain of PABPN1 function relates to OPMD
156
5.4
Future directions
158
5.5
Final conclusions
164
5.6
Figures
165
References
169
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