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The Glycine Arginine Rich Domain of the RNA Binding Protein Nucleolin Regulates its Subcellular Localization

ABSTRACT: Nucleolin is a multifunctional RNA Binding Protein (RBP) with diverse subcellular localizations, including the nucleolus in all eukaryotic cells, the plasma membrane in tumor cells, and the axon in neurons. Here we show that the glycine arginine rich (GAR) domain of nucleolin drives subcellular localization via protein-protein interactions with a kinesin light chain. In addition, GAR sequences mediate plasma membrane interactions of nucleolin. Both these modalities are in addition to the already reported involvement of the GAR domain in liquid-liquid phase separation in the nucleolus. Nucleolin transport to axons requires the GAR domain, and heterozygous GAR deletion mice reveal reduced axonal localization of nucleolin cargo mRNAs and enhanced sensory neuron growth. Thus, the GAR domain governs axonal transport of a growth controlling RNA-RBP complex in neurons, and is a versatile localization determinant for different subcellular compartments. Localization determination by GAR domains may explain why GAR mutants in diverse RBPs are associated with neurodegenerative disease.

SUBMITTER: Dr. Ella Doron-Mandel

PROVIDER: S-SCDT-EMBOJ-2020-107158 | biostudies-other |

REPOSITORIES: biostudies-other

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The glycine arginine-rich domain of the RNA-binding protein nucleolin regulates its subcellular localization.

Project description:Nucleolin is a multifunctional RNA Binding Protein (RBP) with diverse subcellular localizations, including the nucleolus in all eukaryotic cells, the plasma membrane in tumor cells, and the axon in neurons. Here we show that the glycine arginine rich (GAR) domain of nucleolin drives subcellular localization via protein-protein interactions with a kinesin light chain. In addition, GAR sequences mediate plasma membrane interactions of nucleolin. Both these modalities are in addition to the already reported involvement of the GAR domain in liquid-liquid phase separation in the nucleolus. Nucleolin transport to axons requires the GAR domain, and heterozygous GAR deletion mice reveal reduced axonal localization of nucleolin cargo mRNAs and enhanced sensory neuron growth. Thus, the GAR domain governs axonal transport of a growth controlling RNA-RBP complex in neurons, and is a versatile localization determinant for different subcellular compartments. Localization determination by GAR domains may explain why GAR mutants in diverse RBPs are associated with neurodegenerative disease.

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The GAR Domain Determines Subcellular Localization of the Growth Controlling RNA Binding Protein Nucleolin

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GAR-Driven Subcellular Localization of the Growth Controlling RNA Binding Protein Nucleolin

Project description:This SuperSeries is composed of the SubSeries listed below.

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Nucleolin-Mediated RNA Localization Regulates Neuron Growth and Cycling Cell Size.

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

Project description: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. However, 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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