THE REGULATION OF THE SOLUBILITY, OLIGOMERIZATION AND STRUCTURE OF ARGININE-RICH RNA-BINDING PROTEINS BY POST-TRANSLATIONAL MODIFICATION IN ALZHEIMER’S DISEASE Público
Kundinger, Sean (Fall 2021)
Abstract
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
Hypothesis
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
pathogenesis
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
ETD MS
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
Properties
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
Motifs
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
aggregation?
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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