Kundinger, Sean (Fall 2021)

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Post-translational modifications (PTMs) within splicing factor RNA-binding proteins, such as phosphorylation, regulate several critical steps in RNA metabolism including spliceosome assembly, alternative splicing and mRNA export. Previously, the application of conventional mass spectrometry methods have been insufficient to sequence arginine-rich domains of RNA-binding proteins. Here we report a middle-down proteomic approach coupled with electron transfer dissociation (ETD) mass spectrometry to map previously unknown sites of phosphorylation and methylation within the arginine-rich domains of U1-70K, SRSF2 and structurally similar RNA-binding proteins from nuclear extracts of HEK-293 cells. Notably, the arginine-rich LC domains in RNA-binding proteins are densely modified by methylation and phosphorylation compared with the remainder of the proteome, with methylation and phosphorylation favoring RSRS motifs. Analysis of combinatorial PTMs within RSRS motifs indicate that phosphorylation and methylation do not often co-occur, suggesting they may functionally oppose one another. Furthermore, we show that phosphorylation may modify interactions between Arg-rich proteins, as SRSF2 has stronger association with U1-70K and LUC7L3 upon dephosphorylation. We dephosphorylated nuclear extracts in vitro and analyzed equal amounts of detergent-soluble and -insoluble fractions by mass spectrometry-based proteomics. Correlation network analysis resolves 27 distinct modules of differentially soluble nucleoplasm proteins following dephosphorylation. We found classes of RNA-binding proteins with increased aggregation following dephosphorylation, including the SR protein family and the SR-like RBPs although increased aggregation was not observed across broad classes of RBPs. Phosphorylation regulated SRSF2 structure, as native dephosphorylated SRSF2 formed high molecular weight oligomeric species in vitro. Reciprocally, phosphorylation of SRSF2 by serine-/arginine protein kinase 2 (SRPK2) in vitro prevented high molecular weight species formation of SRSF2. Furthermore, SRPK inhibition by SRPIN340 decreased SR protein phosphorylation in vivo and the regulated the cytoplasmic mislocalization of SRSF2 and formation of tubular structures that colocalize with microtubules by immunocytochemical staining. Collectively, these findings demonstrate that phosphorylation is a critical determinant of SR and SR-like protein solubility, oligomerization, and structure. Furthermore, these findings suggest that the level of phosphorylation within arginine-rich domains of RNA-binding proteins may be among the highest in the proteome, and a possible critical suppressor of arginine-rich RNA-binding protein aggregation and mislocalization.

Table of Contents

1.0 RNA-binding protein aggregation in Alzheimer’s disease                                             1                     1.1 An Introduction to Alzheimer’s disease                                                                  1

1.2 A History of Alzheimer’s disease                                                                        1         

                       1.2.1 Pathology of Alzheimer’s disease                                                        3

1.2.2 Genetic heterogeneity of Alzheimer’s disease                                     5

           1.3 Amyloid Cascade Hypothesis                                                                              7

                       1.3.1 Limitations of the Amyloid Cascade                                                    8


           1.4 Links between RNA-binding proteins and                                                          9

neurodegenerative disease

1.4.1 Granules to Fibrils: an equilibrium of                                                  10

Liquid-liquid phase separation (LLPS)

1.4.2 RNA-binding protein pathology in                                                       10

Alzheimer’s disease

1.5 1.5 Using mass spectrometry-based proteomics to                                             12

investigate the pathophysiology of Alzheimer’s disease                                           

1.5.1 Mass spectrometry-based proteomics reveals                                      13

novel protein signatures in Alzheimer’s disease


1.5.2 Identification of U1 small nuclear                                                        13

ribonucleoprotein spliceosome and arginine-rich

RNA-binding proteins enriched in Alzheimer’s

disease insoluble fractions

1.6 The Spliceosome and associated arginine-rich splicing                                      15

factor RNA-binding proteins

1.6.1 Discovery of arginine-rich splicing factor                                            16

RNA-binding proteins           

1.7 Regulation of arginine-rich RNA-binding protein                                              16

function by post-translational modification (PTM)

1.7.1 PTM regulation of spliceosome assembly                                            17

and canonical/alternative splicing

1.7.2 Post-translational modification                                                            18

regulation of RNA-binding protein LLPS

1.7.3 Limitations of using mass                                                                     19

spectrometry-based proteomics to identify PTM

sites in arginine-rich RNA-binding proteins

           1.8 Research Focus and Innovation                                                                           20

           1.9 Figures and Tables                                                                                               21

2.0 Middle-down proteomics reveals dense sites of methylation                                         35

and phosphorylation in arginine-rich RNA-binding proteins

           2.1 Abstract                                                                                                               36

2.2 Introduction                                                                                                         37

           2.3 Materials and Methods                                                                                        42

2.4 Results                                                                                                                 51

           2.4.1 Preparation of Nucleoplasm Fractions                                                 53

Enriched with Arg-rich RBPs

2.4.2 Global Analysis of RNA-binding proteins                                           55

from Nucleoplasm Extracts by Middle-down


2.4.3 Enhanced Sequence Coverage of Arg-rich                                          56

Proteins by Middle-Down ETD MS

2.4.4 Motif Algorithms Resolve RNA-Binding                                            57

Protein Subgroups with Distinct Biological


                       2.4.5 PTM Site Validation                                                                             59

2.4.6 Arg-Rich Domains in RNA-Binding Proteins                                      60

Contain Combinatorial PTMs

2.4.7 Arg-rich Domains in RNA-Binding Proteins                                       61

Are Densely Modified

                       2.4.8 Phosphorylation and Methylation Favor RSRS                                   63


2.4.9 Phosphorylation Regulates Protein-Protein                                          64

Interactions between Structurally Similar Arg-Rich

RNA-Binding Proteins

           2.5 Discussion                                                                                                            65

           2.6 Acknowledgments                                                                                                           70

           2.7 Figures                                                                                                                71

3.0 Phosphorylation regulates arginine-rich RNA-binding protein                                      95

solubility and oligomerization

           3.1 Abstract                                                                                                               96

3.2 Introduction                                                                                                         97

           3.3 Materials and Methods                                                                                        99

3.4 Results                                                                                                                 111

                       3.4.1 Phosphorylation prevents SRSF2 aggregation                                     111

3.4.2 Proteomics reveals RBPs that aggregate                                              112

following dephosphorylation

3.4.3 Arginine-/lysine-rich RNA-binding proteins                                       113

with positive net charge preferentially aggregate

following dephosphorylation

3.4.4 Systems analysis identifies modules of proteins                                  115

with solubility impacted by phosphorylation

3.4.5 Confirmation of hub protein solubility changes                                   116

following dephosphorylation

3.4.6 Phosphorylation decreases NCPR and regulates                                  117

the oligomerization of arginine-rich SRSF2

3.4.7 SRPK inhibitor SRPIN340 decreases SR protein                                118

phosphorylation and increases SRSF2 granule and

tubule formation

3.4.8 SRSF2 interacts with microtubule subunit                                           120

proteins α- and β-tubulin        

           3.5 Discussion                                                                                                            121

           3.6 Acknowledgments                                                                                                           123

           3.7 Figures                                                                                                                 125

4.0 Discussion                                                                                                                        147

4.1 Summary                                                                                                              147

4.2 Future directions of mass spectrometry to improve                                            147

sequencing of arginine-rich RNA-binding proteins

4.3 Use of middle-down proteomic approaches to sequence                                    149

post-translational modification sites in arginine-rich

RNA-binding proteins in Alzheimer’s disease

4.4 Recent discoveries of RNA-binding protein functions                                       149

regulated by post-translational modifications

4.5 Is phosphorylation a trigger or a consequence of protein                                   150


4.6 Does phosphorylation regulate the core splicing function                                  152

of arginine-rich RNA-binding proteins?

4.7 The in vivo roles of phosphatases                                                                        153

4.8 Implications of phosphorylation dysregulation in AD                                        154

4.9 Future Directions                                                                                                 155

4.10 Figures                                                                                                               158

5.0 References                                                                                                                       160

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