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bioRxiv · 10.1101/2021.06.30.450578

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

Kundinger, S. R.·Dammer, E. B.·Yin, L.·Hurst, C.·Ping, L.·Duong, D.·Seyfried, N.
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Abstract

Post-translational modifications (PTMs) within splicing factor RNA-binding proteins (RBPs), such as phosphorylation, regulate several critical steps in RNA metabolism including spliceosome assembly, alternative splicing and mRNA export. Notably, the arginine-/serine-rich (RS) domains in SR proteins are densely modified by phosphorylation compared with the remainder of the proteome. Previously, we showed that dephosphorylation of SRSF2 regulated increased interactions with similar arginine-rich RBPs U1-70K and LUC7L3. 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 to 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. 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 prevented high molecular weight SRSF2 species formation. Furthermore, we pharmacologically inhibited SRPKs in mammalian cells and observed increased cytoplasmic granules as well as the formation of cytoplasmic SRSF2 tubular structures that associate with microtubules by immunocytochemical staining. Collectively, these findings demonstrate that phosphorylation may be a critical modification that prevents arginine-rich RBP insolubility and oligomerization.

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Kundinger, S. R., Dammer, E. B., Yin, L., Hurst, C., Ping, L., Duong, D., Seyfried, N.. 2021-06-30. Phosphorylation regulates arginine-rich RNA-binding protein solubility and oligomerization. https://doi.org/10.1101/2021.06.30.450578

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