Phosphorylation regulates arginine-rich RNA-binding protein solubility and oligomerization
| Autor: | Lingyan Ping, Duc M. Duong, Sohail Khoshnevis, Sean R. Kundinger, Sarah Shapley, Cheyenne Hurst, Eric B. Dammer, Luming Yin, Nicholas T. Seyfried, Homa Ghalei |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
SC35
FDR false discovery rate WGCNA weighted gene correlation network analysis Phosphatase RNA-binding proteins RNA-binding protein Protein Serine-Threonine Kinases protein interactions Biochemistry LLPS liquid–liquid phase-separated Ribonucleoprotein U1 Small Nuclear TFA trifluoroacetic acid Dephosphorylation SR protein Humans Phosphorylation Protein kinase A posttranslational modifications (PTMs) CIP calf intestinal alkaline phosphatase LC-MS/MS liquid chromatography coupled with tandem mass spectrometry Molecular Biology ABC ammonium bicarbonate mass spectrometry FA formic acid Nucleoplasm Serine-Arginine Splicing Factors Protein Stability Chemistry NCPR net charge per reside Alternative splicing Nuclear Proteins Cell Biology Cell biology SRSF2 RRM RNA recognition motif HEK293 Cells LC low complexity SRPK SR protein kinase PTM posttranslational modification RBP RNA-binding protein Protein Multimerization Research Article |
| Zdroj: | The Journal of Biological Chemistry |
| ISSN: | 0021-9258 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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