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.
Subject(s)
Cell Nucleolus/metabolism , Ganglia, Spinal/metabolism , Kinesins/metabolism , Neurons/metabolism , Phosphoproteins/chemistry , RNA-Binding Proteins/chemistry , Sciatic Nerve/metabolism , Amino Acid Sequence , Animals , Axonal Transport/genetics , Cell Line, Tumor , Cell Nucleolus/ultrastructure , Ganglia, Spinal/cytology , Gene Expression , HEK293 Cells , HeLa Cells , Humans , Kinesins/genetics , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mutation , Neurons/cytology , Phosphoproteins/genetics , Phosphoproteins/metabolism , Primary Cell Culture , Protein Domains , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Sciatic Nerve/cytology , NucleolinABSTRACT
Importin ß1 (KPNB1) is a nucleocytoplasmic transport factor with critical roles in both cytoplasmic and nucleocytoplasmic transport, hence there is keen interest in the characterization of its subcellular interactomes. We found limited efficiency of BioID in the detection of importin complex cargos and therefore generated a highly specific and sensitive anti-KPNB1 monoclonal antibody to enable biotinylation by antibody recognition analysis of importin ß1 interactomes. The monoclonal antibody recognizes an epitope comprising residues 301-320 of human KPBN1 and strikingly is highly specific for cytoplasmic KPNB1 in diverse applications, with little reaction with KPNB1 in the nucleus. Biotinylation by antibody recognition with this novel antibody revealed numerous new interactors of importin ß1, expanding the KPNB1 interactome to cytoplasmic and signaling complexes that highlight potential new functions for the importins complex beyond nucleocytoplasmic transport. Data are available via ProteomeXchange with identifier PXD032728.
Subject(s)
Antibodies, Monoclonal , Karyopherins , Humans , Karyopherins/metabolism , Antibodies, Monoclonal/metabolism , beta Karyopherins/metabolism , Active Transport, Cell Nucleus , Cytoplasm/metabolism , Cell Nucleus/metabolismABSTRACT
How is neuropathic pain regulated in peripheral sensory neurons? Importins are key regulators of nucleocytoplasmic transport. In this study, we found that importin α3 (also known as karyopherin subunit alpha 4) can control pain responsiveness in peripheral sensory neurons in mice. Importin α3 knockout or sensory neuron-specific knockdown in mice reduced responsiveness to diverse noxious stimuli and increased tolerance to neuropathic pain. Importin α3-bound c-Fos and importin α3-deficient neurons were impaired in c-Fos nuclear import. Knockdown or dominant-negative inhibition of c-Fos or c-Jun in sensory neurons reduced neuropathic pain. In silico screens identified drugs that mimic importin α3 deficiency. These drugs attenuated neuropathic pain and reduced c-Fos nuclear localization. Thus, perturbing c-Fos nuclear import by importin α3 in peripheral neurons can promote analgesia.
Subject(s)
Chronic Pain/physiopathology , Neuralgia/physiopathology , Sensory Receptor Cells/physiology , alpha Karyopherins/physiology , Active Transport, Cell Nucleus , Animals , Benzophenones/pharmacology , Chronic Pain/genetics , Gene Expression Profiling , Gene Knockdown Techniques , Isoxazoles/pharmacology , Mice , Mice, Inbred C57BL , Neuralgia/genetics , Proto-Oncogene Proteins c-fos/antagonists & inhibitors , Proto-Oncogene Proteins c-fos/metabolism , Transcription Factor AP-1/metabolism , alpha Karyopherins/geneticsABSTRACT
Importins mediate transport from synapse to soma and from cytoplasm to nucleus, suggesting that perturbation of importin-dependent pathways should have significant neuronal consequences. A behavioral screen on five importin α knockout lines revealed that reduced expression of importin α5 (KPNA1) in hippocampal neurons specifically decreases anxiety in mice. Re-expression of importin α5 in ventral hippocampus of knockout animals increased anxiety behaviors to wild-type levels. Hippocampal neurons lacking importin α5 reveal changes in presynaptic plasticity and modified expression of MeCP2-regulated genes, including sphingosine kinase 1 (Sphk1). Knockout of importin α5, but not importin α3 or α4, reduces MeCP2 nuclear localization in hippocampal neurons. A Sphk1 blocker reverses anxiolysis in the importin α5 knockout mouse, while pharmacological activation of sphingosine signaling has robust anxiolytic effects in wild-type animals. Thus, importin α5 influences sphingosine-sensitive anxiety pathways by regulating MeCP2 nuclear import in hippocampal neurons.