RNA-binding proteins with mixed charge domains self-assemble, aggregate, and interact with core pathologies in Alzheimer's Disease Público

Abstract

U1-70K and other RNA binding proteins (RBPs) are mislocalized to cytoplasmic neurofibrillary Tau aggregates in Alzheimer’s disease (AD), yet the mechanisms that cause their aggregation are incompletely understood. Many RBPs that aggregate in neurodegenerative diseases self-assemble into RNA granules through intrinsically disordered low complexity (LC) domains. We report here that a LC domain within U1-70K of mixed charge, containing highly repetitive complementary repeats of basic (R/K) and acidic (D/E) residues, shares many of the same properties of the Q/N-rich LC domains found in the RBPs TDP-43 and FUS. These properties include the ability to self-assemble into oligomers, and to form nuclear granules. To analyze the functional roles of the U1-70K LC domains, we performed co-immunoprecipitation and quantitative mass spectrometry analysis of recombinant U1-70K and deletions lacking the C-terminal LC domain(s). A network-driven approach resolved functional classes of U1-70K interacting proteins that showed dependency on the U1-70K LC domain(s) for their interaction. This included structurally similar RBPs, such as LUC7L3 and RBM25, which require their respective mixed charge domains for reciprocal interactions with U1-70K and for participation in nuclear RNA granules. Strikingly, a significant proportion of RBPs with mixed charge domains have elevated insolubility in the AD brain proteome compared to controls. Furthermore, we show that the mixed charge LC domain of U1-70K can interact with β-amyloid and Tau from AD brain. This supports a hypothesis that β-amyloid directly or indirectly mediates interactions between mixed charge structural motifs on U1-70K and related RBPs with pathological Tau in AD.

Table of Contents

Abbreviations                                                                          8

Chapter 1: Introduction

1.1 A Brief History of Alzheimer's Disease Research                  10

1.1.1 Neuropathology of Alzheimer's Disease                              10

1.1.2 Genetics of Alzheimer's Disease                                          12

1.2 Amyloid Hypothesis                                                                    13

1.2.1 Limitations of the Amyloid Hypothesis                                13

1.3 Alzheimer's Disease is a continuum                                          15

1.4 Proteomics as a tool to understand biology                            16

1.4.1 Proteomics analysis of detergent-insoluble proteome across neurodegenerative disease reveals U1snRNP aggregation specially in AD brain                                                                                       17

1.4.2 U1snRNP Function                                                                   18

1.4.3 Other links between the U1snRNP and AD                         19

1.5 Links between RNA granules and neurodegenerative disease  19

1.5.1 RNA Granules                                                                           20

1.5.2 Biophysical properties of RNA granules                              21

1.5.3 Tau, LLPS and RNA binding proteins                                   22

1.6 Properties of U1-70K aggregation                                            23

1.6.1 Special Characteristics of the U1-70K LC1 domain           24

1.7 Research focus and Innovation                                                 25

1.8 Figures                                                                                          26

Chapter 2: RNA-binding proteins with mixed charge domains self-assemble and aggregate in Alzheimer's Disease

2.1 Abstract                                                                                         32

2.2 Introduction                                                                                  33

2.3 Results                                                                                            36

2.3.1 The LC1 domain of U1-70K is necessary and sufficient for self-association in cells    36

2.3.2 The U1-70K LC1 domain oligomerizes in vitro                    37

2.3.3 The U1-70K LC1 domain is necessary and sufficient for robust nuclear granule localization      38

2.3.4 Protein-Protein interaction network analysis resolves functionally distinct classes of U1-70K interacting proteins         38

2.3.5 Confirmation of U1-70K LC1 dependent interacting proteins   40

2.3.6 The mRNA processing module is enriched with structurally similar      41

 RNA-binding proteins harboring mixed charge domains                      42

2.3.7 Mixed charge domains in LUC7L3 and RBM25 are necessary for reciprocal interactions with U1-70K and nuclear RNA granule assembly       43

2.3.8 RNA binding proteins with mixed charge domains have enhanced insolubility in AD brain      44

2.3.9 The LC1 domain of U1-70K interacts with pathological Tau from AD brain     45

2.4 Discussion                                                                                                47

2.5 Materials and Methods                                                                          48

2.6 Acknowledgement                                                                                  57

2.7 Tables and Figures                                                                                  58

Chapter 3: Discussion                                                                       72

3.1 Biological function of Mixed Charge domains                                   72

3.1.1 Parallels between RNA binding protein aggregation events in AD and ALS   72

3.1.2 Implication of U1-70K interactions with Aβ and Tau                74

3.1.3 Implication of U1-70K interactions with Aβ and Tau                75

3.2 A Comprehensive Model for AD Pathogenesis: A role for mixed charge RNA binding proteins        77

3.3 Power of Network Approaches                                                            78

3.4 Limitations                                                                                              78

3.5 Remaining Questions and Future Studies                                         79

3.5.1 Aβ specificity                                                                            79

3.5.2 Correlating Aβ-LC1 Binding to Disease Progression                  80

3.5.3 Determining the Biological Purpose of U1-70K-Ribosome Interactions   80

3.5.4 Examining Biophysical Properties of LC1 U1-70K LLPS             81

3.6 Long Term Future Directions: Factors that regulate RNA Granule disassembly are viable targets for therapeutics  82

Figures                                                                                                            84

4.0 References                                                                                             85

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Biochemistry, Cell & Developmental Biology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Neuroscience Biology, Cell Biology, Bioinformatics
Palavra-chave	U1-70K RBM25 Basic Acidic Dipeptide domain LUC7L3 Alzheimer's Disease BAD protein
Committee Chair / Thesis Advisor	Seyfried, Nicholas, Emory University
Committee Members	Levey, Allan, Emory University Reines, Daniel, Emory University Bassell, Gary, Emory University Corbett, Anita, Emory University

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