Phosphorylation regulates arginine-rich RNA-binding protein solubility and oligomerization

Posttranslational modifications (PTMs) such as phosphorylation of RNA-binding proteins (RBPs) regulate several critical steps in RNA metabolism, including spliceosome assembly, alternative splicing, and mRNA export. Notably, serine-/arginine- (SR)-rich RBPs are densely phosphorylated compared with the remainder of the proteome. Previously, we showed that dephosphorylation of the splicing factor SRSF2 regulated increased interactions with similar arginine-rich RBPs U1-70K and LUC7L3. However, the large-scale functional and structural impact of these modifications on RBPs remains unclear. In this work, we dephosphorylated nuclear extracts using phosphatase in vitro and analyzed equal amounts of detergent-soluble and -insoluble fractions by mass-spectrometry-based proteomics. Correlation network analysis resolved 27 distinct modules of differentially soluble nucleoplasm proteins. We found classes of arginine-rich RBPs that decrease in solubility following dephosphorylation and enrich the insoluble pelleted fraction, including the SR protein family and the SR-like LUC7L RBP family. Importantly, increased insolubility was not observed across broad classes of RBPs. We determined that phosphorylation regulated SRSF2 structure, as dephosphorylated SRSF2 formed high-molecular-weight oligomeric species in vitro. Reciprocally, phosphorylation of SRSF2 by serine/arginine protein kinase 2 (SRPK2) in vitro decreased high-molecular-weight SRSF2 species formation. Furthermore, upon pharmacological inhibition of SRPKs in mammalian cells, we observed SRSF2 cytoplasmic mislocalization and increased formation of cytoplasmic granules as well as cytoplasmic tubular structures that associated with microtubules by immunocytochemical staining. Collectively, these findings demonstrate that phosphorylation may be a critical modification that prevents arginine-rich RBP insolubility and oligomerization. Posttranslational modifications (PTMs) such as phosphorylation of RNA-binding proteins (RBPs) regulate several critical steps in RNA metabolism, including spliceosome assembly, alternative splicing, and mRNA export. Notably, serine-/arginine- (SR)-rich RBPs are densely phosphorylated compared with the remainder of the proteome. Previously, we showed that dephosphorylation of the splicing factor SRSF2 regulated increased interactions with similar arginine-rich RBPs U1-70K and LUC7L3. However, the large-scale functional and structural impact of these modifications on RBPs remains unclear. In this work, we dephosphorylated nuclear extracts using phosphatase in vitro and analyzed equal amounts of detergent-soluble and -insoluble fractions by mass-spectrometry-based proteomics. Correlation network analysis resolved 27 distinct modules of differentially soluble nucleoplasm proteins. We found classes of arginine-rich RBPs that decrease in solubility following dephosphorylation and enrich the insoluble pelleted fraction, including the SR protein family and the SR-like LUC7L RBP family. Importantly, increased insolubility was not observed across broad classes of RBPs. We determined that phosphorylation regulated SRSF2 structure, as dephosphorylated SRSF2 formed high-molecular-weight oligomeric species in vitro. Reciprocally, phosphorylation of SRSF2 by serine/arginine protein kinase 2 (SRPK2) in vitro decreased high-molecular-weight SRSF2 species formation. Furthermore, upon pharmacological inhibition of SRPKs in mammalian cells, we observed SRSF2 cytoplasmic mislocalization and increased formation of cytoplasmic granules as well as cytoplasmic tubular structures that associated with microtubules by immunocytochemical staining. Collectively, these findings demonstrate that phosphorylation may be a critical modification that prevents arginine-rich RBP insolubility and oligomerization. RNA-binding proteins (RBPs) cooperatively engage both RNA and protein (1Gebauer F. Schwarzl T. Valcárcel J. Hentze M.W. RNA-binding proteins in human genetic disease.Nat. Rev. Genet. 2021; 22: 185-198Crossref PubMed Scopus (90) Google Scholar). RBPs frequently contain an RNA-binding domain, typically K-homology (KH), or RNA recognition motif (RRM) domains, which allow the RBP to achieve sequence-specific binding to target RNA molecules (2Hentze M.W. Castello A. Schwarzl T. Preiss T. A brave new world of RNA-binding proteins.Nat. Rev. Mol. Cell Biol. 2018; 19: 327-341Crossref PubMed Scopus (607) Google Scholar). Unbiased RNA interactome studies have identified many RBPs containing low-complexity (LC) domains that participate in both RNA and protein interactions (2Hentze M.W. Castello A. Schwarzl T. Preiss T. 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Collectively, this work reinforces phosphorylation as an important regulator of SR protein solubility and structure and suggests that phosphorylation may be a preventative cellular mechanism against arginine-rich RBP aggregation. Here, we use SRSF2 as a paradigm to study the regulation of arginine-rich RBP solubility, structure, and morphology by phosphorylation. In SRSF2, the arginine-/serine-rich (RS) domain is highly phosphorylated (28Kundinger S.R. Bishof I. Dammer E.B. Duong D.M. Seyfried N.T. Middle-down proteomics reveals dense sites of methylation and phosphorylation in arginine-rich RNA-binding proteins.J. Proteome Res. 2020; 19: 1574-1591Crossref PubMed Scopus (7) Google Scholar), a region with high probability of intrinsic disorder (Fig. S1A) (49Jones D.T. Cozzetto D. DISOPRED3: Precise disordered region predictions with annotated protein-binding activity.Bioinformatics. 2015; 31: 857-863Crossref PubMed Scopus (455) Google Scholar, 50Haynes C. Iakoucheva L.M. Serine/arginine-rich splicing factors belong to a class of intrinsically disordered proteins.Nucleic Acids Res. 2006; 34: 305-312Crossref PubMed Scopus (80) Google Scholar). We incubated lysates containing recombinant SRSF2-myc, a known phosphoprotein, with CIP, which corresponded to a lower-molecular-weight SRSF2 band (Fig. S1B) by SDS-PAGE, suggesting substantial dephosphorylation of SRSF2 (51Keshwani M.M. Aubol B.E. Fattet L. Ma C.T. Qiu J. Jennings P.A. Fu X.D. Adams J.A. Conserved proline-directed phosphorylation regulates SR protein conformation and splicing function.Biochem. J. 2015; 466: 311-322Crossref PubMed Scopus (28) Google Scholar, 52Aubol B.E. Wu G. Keshwani M.M. Movassat M. Fattet L. Hertel K.J. Fu X.D. Adams J.A. Release of SR proteins from CLK1 by SRPK1: A symbiotic kinase system for phosphorylation control of pre-mRNA splicing.Mol. Cell. 2016; 63: 218-228Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). 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Chem. 2015; 290: 30931-30946Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar) (Fig. S1C) and again observed increased migration of SRSF2. Furthermore, we saw an increase in hypoSRSF2 immunoreactivity, demonstrating that SRSF2 is indeed dephosphorylated. We then asked whether phosphorylation regulates the solubility of SRSF2. Detergent-soluble (S) and -insoluble pelleted (P) fractions were isolated following mock (−CIP) or phosphatase (+CIP) treatment (Fig. 1A). We resolved equal amounts of total (T), soluble (S), and pelleted (P) fractions by SDS-PAGE and immunoblotted for SRSF2-myc (Fig. 1B). Labeling of each fraction with the hypoSRSF2 antibody confirmed that the pellet fraction was enriched with hypoSRSF2 species (Fig. S1D). While phosphorylated SRSF2-myc was primarily soluble (68% of total) in the mock condition, dephosphorylated SRSF2-myc significantly decreased in solubility, enriching to the detergent-insoluble pellet fraction (89% of total, Fig. 1C). This suggests that in addition to SRSF2, similar arginine-rich RBPs or groups of RBPs may experience altered solubility following dephosphorylation. We next sought to globally identify and quantify RBPs that aggregate following dephosphorylation. Following dephosphorylation, the soluble and insoluble (i.e., pellet) samples were analyzed by label-free quantitative proteomics, using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) in biological quadruplicate (Fig. 1A, Table S1). Notably, dephosphorylation did not induce global aggregation of the nuclear proteome, as insoluble pellet fraction protein concentrations were unchanged after phosphatase incubation (Table S2). Following database search and removal of proteins with >50% missing values and single peptide identifications, we identified 4120 unique proteins (Table S3 and S4). To discover proteins with the largest change in solubility following dephosphorylation, we calculated the log2 fold differences of fraction insoluble values between phosphatase and mock treatments and visualized this as a volcano plot (Fig. 1D, Tables S4 and S5). Proteins were highlighted with increased or decreased insolubility if at least a twofold increase or decrease in fraction insoluble values, respectively, was observed with a p value less than 0.05 (two-tailed paired t test). Relatively few proteins (n = 10) enriched to the soluble fraction following dephosphorylation. In contrast, many more proteins (n = 734) experienced increased aggregation following dephosphorylation. To ask whether serine-/arginine-rich (SR) proteins were enriched within this group, we performed a homology search of the RS domain of SRSF2 using the protein BLAST tool. We identified several proteins with high homology to the RS domain of SRSF2 (n = 193), including other SR proteins (SRSF3/4/5/6/7), as well as the SR-like proteins SRRM1 and LUC7L3 (Fig. S2A, Table S6). Using a one-dimensional hypergeometric Fisher’s exact test (FET) analysis, we concluded that the SR/SR-like group was significantly enriched to the list of proteins that experienced significantly decreased solubility following dephosphorylation (BH-corrected p value = 0.0132) (Fig. S2B). These observations suggest that phosphorylation is an important PTM that regulates the solubility of SRSF2, as well as the solubilities of similar arginine-/serine-rich RBPs. Phosphorylation significantly alt