Background:The mechanisms underlying U1-70K aggregation in AD are unknown. Results: AD brain homogenates can induce the aggregation of soluble U1-70K, and disordered low complexity domains are necessary for U1-70K aggregation. Conclusion: U1-70K in AD may directly sequester normal soluble forms of U1-70K into insoluble aggregates. Significance: These observations highlight the importance of low complexity domains in RNA-binding protein aggregation in neurodegenerative disease.

The U1 small nuclear ribonucleoprotein 70 kDa (U1-70K) and other RNA-binding proteins (RBPs) are mislocalized to cytoplasmic neurofibrillary Tau aggregates in Alzheimer's disease (AD), yet the co-aggregation mechanisms are incompletely understood. U1-70K harbors two disordered low-complexity domains (LC1 and LC2) that are necessary for aggregation in AD brain extracts. The LC1 domain contains highly repetitive basic (Arg/Lys) and acidic (Asp/Glu) residues, referred to as a basic-acidic dipeptide (BAD) domain. We report here that this domain shares many of the properties of the Gln/Asn-rich LC domains in RBPs that also aggregate in neurodegenerative disease. These properties included self-assembly into oligomers and localization to nuclear granules. Co-immunoprecipitations of recombinant U1-70K and deletions lacking the LC domain(s) followed by quantitative proteomic analyses were used to resolve functional classes of U1-70K-interacting proteins that depend on the BAD domain for their interaction. Within this interaction network, we identified a class of RBPs with BAD domains nearly identical to that found in U1-70K. Two members of this class, LUC7L3 and RBM25, required their respective BAD domains for reciprocal interactions with U1-70K and nuclear granule localization. Strikingly, a significant proportion of RBPs with BAD domains had elevated insolubility in the AD brain proteome. Furthermore, we show that the BAD domain of U1-70K can interact with Tau from AD brains but not from other tauopathies. These findings highlight a mechanistic role for BAD domains in stabilizing RBP interactions and in potentially mediating co-aggregation with the pathological AD-specific Tau isoforms.

Middle-down proteomics reveals arginine-rich RNA-binding proteins contain many sites of methylation and phosphorylation..

We have used mass spectrometry (MS) to characterize protein signaling in lipopolysaccharide (LPS)-stimulated macrophages from human blood, human THP1 cells, mouse bone marrow, and mouse Raw264.7 cells. Protein ADP-ribosylation was truncated down to phosphoribose, allowing for enrichment and identification of the resulting phosphoribosylated peptides alongside phosphopeptides. Size exclusion chromatography-MS (SEC-MS) was used to separate proteoforms by size; protein complexes were then identified by weighted correlation network analysis (WGCNA) based on their correlated movement into or out of SEC fractions following stimulation, presenting an analysis method for SEC-MS that does not rely on established databases. We highlight two modules of interest: one linked to the apoptosis signal-regulating kinase (ASK) signalosome and the other containing poly(ADP-ribose) polymerase 9 (PARP9). Finally, PARP inhibition was used to perturb the characterized systems, demonstrating the importance of ADP-ribosylation for the global interactome. All post-translational modification (PTM) and interactome data have been aggregated into a meta-database of 6729 proteins, with ADP-ribosylation characterized on 2905 proteins and phosphorylation characterized on 2669 proteins. This databasetitled MAPCD, for Macrophage ADP-ribosylation, Phosphorylation, and Complex Dynamicsserves as an invaluable resource for studying crosstalk between the ADP-ribosylome, phosphoproteome, and interactome.

The RNA binding protein U1‐70K aggregates via an unknown mechanism in Alzheimer's disease (AD). Interestingly, AD brain homogenates can induce the aggregation of soluble U1‐70K from control brain or recombinant U1‐70K (rU1‐70K), rendering it detergent‐insoluble. The intrinsically disordered C‐terminus of U1‐70K is necessary for aggregation and harbors two low‐complexity (LC) domains, LC1 and LC2. Similar LC domains have been shown to polymerize into amyloid‐like aggregates in vitro, and the RNA binding proteins TDP‐43 and FUS, which also harbor LC domains, form detergent‐insoluble amyloid‐like aggregates in neurodegenerative disease. Our objective is to determine if the LC domains of U1‐70K are necessary for self‐association. Addressing this question may provide mechanistic insight into the aggregation of U1‐70K in AD. To investigate this question we performed co‐immunoprecipitations of full‐length rU1‐70K and various mutants lacking either the LC1, LC2, or both LC domains. These assays revealed that the LC1 domain (amino acids 231‐308) of rU1‐70K is necessary and sufficient for association with native U1‐70K. Similarly, we utilized bio‐layer interferometry to measure the binding affinity of the LC1 domain with a synthetic peptide corresponding to amino acids 252‐270 of U1‐70K. These data provide evidence for a direct self‐association between regions of the LC1 domain. Finally, by immunocytochemistry we show that rU1‐70K mutants lacking the LC1 domain have an impaired ability to form nuclear granule structures. Together these data indicate that the disordered LC1 domain is necessary for U1‐70K self‐association and subnuclear compartmentalization.Sources of Research Support:NIRG‐12‐242297 (NTS) and T32‐NS007480 (IB)

Abbreviations:MS: mass spectrometry ETD: electron transfer dissociation PTM: post-translational modification AD: Alzheimer's disease LC: liquid chromatography RBP: RNA-binding proteins PSM : peptide spectral match Briefs:Middle-down proteomics reveals arginine-rich RNA-binding proteins contain many sites of methylation and phosphorylation. AbstractArginine (Arg)-rich RNA-binding proteins play an integral role in RNA metabolism. Posttranslational modifications (PTMs) within Arg-rich domains, such as phosphorylation and methylation, regulate multiple steps in RNA metabolism. However, the identification of PTMs within Arg-rich domains with complete trypsin digestion is extremely challenging due to the high density of Arg residues within these 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 Arg-rich domains of U1-70K and structurally similar RNA-binding proteins from nuclear extracts of HEK293 cells. Remarkably, the Arg-rich domains in RNA-binding proteins are densely modified by methylation and phosphorylation compared with the remainder of the proteome, with di-methylation and phosphorylation favoring RSRS motifs. Although they favor a common motif, analysis of combinatorial PTMs within RSRS motifs indicate that phosphorylation and methylation do not often co-occur, suggesting they may functionally oppose one another. Collectively, these findings suggest that the level of PTMs within Arg-rich domains may be among the highest in the proteome, and a possible unexplored regulator of RNA metabolism. These data also serve as a resource to facilitate future mechanistic studies of the role of PTMs in RNA-binding protein structure and function.Key proteins that carry out specialized biological processes such as RNA splicing, polyadenylation and transport, contain domains disproportionately enriched with arginine [1,2].RNA-binding proteins (RBPs) that harbor Arginine (Arg)-rich domains may be broadly classified into two subsets based on residue composition. One class of these RBPs contains highly repetitive complementary repeats of basic (K/R) and acidic (D/E) residues, that we have previously referred to as Basic Acidic Dipeptide (BAD) domains [3]. Importantly, BAD domains facilitate protein aggregation, and in the context of Alzheimer's disease (AD), facilitate interactions with pathological Tau protein [3]. A second subset, related to the BAD proteins, are the Arginine/serinerich (RS) domains that are ubiquitous in the Serine/Arginine (SR) family of proteins [2]. Upon serine phosphorylation, RS domains mimic BAD domains with a similarly alternating basic-acidic dipeptide sequence pattern. The RS domains are commonly found in splicing factors and are essential for alternative splicing, protein-protein interactions, and localization [4-9]. The functional and structural diversity of the proteome is markedly increased through posttranslational modifications (PTMs), which...

may accelerate or exacerbate disease pathogenesis 1 . b-wrapin proteins were engineered to bind and sequester amyloidogenic monomers, and thus prohibit amyloid formation. ZAb 3 , the first reported b-wrapin 2 sequesters a b-hairpin conformation of Ab, prohibiting aggregation of Ab monomers into toxic forms 3 . b-wrapin variants have been engineered with varying activities for a-syn and IAPP [4][5][6] , and ZSYM73, a b-wrapin with a pM affinity to Ab 7 . Methods: Molecular dynamics simulations, free energy calculations, and surface plasmon resonance, among others enable us to uncover the binding and specificity of b-wrapins' for the three amyloidogenic proteins 8 . Results:Our studies reveal the key interactions acting as potential switches diminishing b-wrapins' affinity for Ab/ a-syn, and suggest that IAPP is a comparatively promiscuous bwrapin target. We delineate the distinct role of energetic driving determinants leading to b-wrapin binding and specificity; while both nonpolar and polar interactions synergistically contribute to binding, the polar binding energy component is the key energetic determinant contributing to b-wrapins' high-affinity. In line with this, our studies show that the high-affinity of ZSYM73 7 is attributed to salt-bridges stabilizing the ZSYM73:Ab complex. Additionally we show that multi-targeted binding properties of b-wrapins originate mainly from optimized interactions between b-wrapin residues and sets of residues in the three amyloidogenic proteins with similar physicochemical properties. Conclusions: Computational tools allow us to predict b-wrapin affinity for amyloidogenic proteins. We are currently using the insights from our studies to design new b-wrapins with improved affinities for one or combinations of the amyloidogenic proteins, which may constitute a promising and efficient direction for the future treatment of Alzheimer's disease in place of molecules binding to Ab only. 1

U1 small nuclear ribonucleoprotein 70 kDa (U1-70K) and other RNA binding proteins (RBPs) are mislocalized to cytoplasmic neurofibrillary Tau aggregates in Alzheimer's disease (AD), yet understanding of the mechanisms that cause their aggregation is limited. 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 coimmunoprecipitation 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 AD brain proteome compared to controls. Furthermore, we show that the mixed charge LC domain of U1-70K can interact with Tau from AD brain. These findings highlight mechanisms for mixed charge domains in stabilizing RBP interactions and in potentially mediating coaggregation with pathological Tau isoforms in AD. INTRODUCTION The molecular processes that contribute to neurodegenerative diseases are not well understood. Recent observations suggest that numerous neurodegenerative diseases are promoted by the accumulation of RNA-binding protein (RBP) aggregates (1-3). This includes Alzheimer's disease (AD), where pathological RNA-protein aggregates are often, but not exclusively, associated with Tau neurofibrillary . CC-BY 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/243014 doi: bioRxiv preprint first posted online Jan. 4, 2018; Mixed Charge RNA binding proteins aggregate in AD 2 tangles in brain (4). For example, U1 small nuclear ribonucleoprotein 70 kDa (U1-70K) and other core components of the spliceosome complex form detergent-insoluble aggregates in both sporadic and familial human cases of AD (5-7). Furthermore, RNA-seq analysis from AD and control brains revealed a significant accumulation of unspliced pre-mRNA disease related transcripts in AD consistent with a loss of U1-spliceosome function (7,8). Currently, our knowledge of the specific mechanisms underlying U1-70K aggregation is limited. This has proved to be a barrier to developing cellular models that wou...