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

https://doi.org/10.1016/j.jbc.2021.101306Get rights and content
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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.

Keywords

RNA-binding proteins
phosphorylation
posttranslational modifications (PTMs)
mass spectrometry
protein interactions
SRSF2
SC35

Abbreviations

ABC
ammonium bicarbonate
CIP
calf intestinal alkaline phosphatase
FA
formic acid
FDR
false discovery rate
LC
low complexity
LC-MS/MS
liquid chromatography coupled with tandem mass spectrometry
LLPS
liquid–liquid phase-separated
NCPR
net charge per reside
PTM
posttranslational modification
RBP
RNA-binding protein
RRM
RNA recognition motif
SRPK
SR protein kinase
TFA
trifluoroacetic acid
WGCNA
weighted gene correlation network analysis

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