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

Isaac Bishof (Summer 2018)

Permanent URL: https://etd.library.emory.edu/concern/etds/5x21tf51p?locale=en


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

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

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