title: Immunoprecipitation and mass spectrometry defines an extensive RBM45 protein-protein interaction network.
abstract: The pathological accumulation of RNA-binding proteins (RBPs) within inclusion bodies is a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). RBP aggregation results in both toxic gain and loss of normal function. Determining the protein binding partners and normal functions of disease-associated RBPs is necessary to fully understand molecular mechanisms of RBPs in disease. Herein, we characterized the protein-protein interactions (PPIs) of RBM45, a RBP that localizes to inclusions in ALS/FTLD. Using immunoprecipitation coupled to mass spectrometry (IP-MS), we identified 132 proteins that specifically interact with RBM45 within HEK293 cells. Select PPIs were validated by immunoblot and immunocytochemistry, demonstrating that RBM45 associates with a number of other RBPs primarily via RNA-dependent interactions in the nucleus. Analysis of the biological processes and pathways associated with RBM45-interacting proteins indicates enrichment for nuclear RNA processing/splicing via association with hnRNP proteins and cytoplasmic RNA translation via eiF2 and eiF4 pathways. Moreover, several other ALS-linked RBPs, including TDP-43, FUS, Matrin-3, and hnRNP-A1, interact with RBM45, consistent with prior observations of these proteins within intracellular inclusions in ALS/FTLD. Taken together, our results define a PPI network for RBM45, suggest novel functions for this protein, and provide new insights into the contributions of RBM45 to neurodegeneration in ALS/FTLD. This article is part of a Special Issue entitled SI:RNA Metabolism in Disease.
authors.affiliation: Divisions of Neurology and Neurobiology, Barrow Neurological Institute, St. Joseph׳s Hospital and Medical Center, Phoenix, AZ 85013, USA.
authors.forename: Yang
authors.initials: Y
authors.lastname: Li
authors.affiliation: Divisions of Neurology and Neurobiology, Barrow Neurological Institute, St. Joseph׳s Hospital and Medical Center, Phoenix, AZ 85013, USA; University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
authors.forename: Mahlon
authors.initials: M
authors.lastname: Collins
authors.affiliation: Divisions of Neurology and Neurobiology, Barrow Neurological Institute, St. Joseph׳s Hospital and Medical Center, Phoenix, AZ 85013, USA.
authors.forename: Jiyan
authors.initials: J
authors.lastname: An
authors.affiliation: Divisions of Neurology and Neurobiology, Barrow Neurological Institute, St. Joseph׳s Hospital and Medical Center, Phoenix, AZ 85013, USA.
authors.forename: Rachel
authors.initials: R
authors.lastname: Geiser
authors.affiliation: Center for Proteomics, TGen (Translational Genomics Research Institute), Phoenix, AZ 85004, USA.
authors.forename: Tony
authors.initials: T
authors.lastname: Tegeler
authors.affiliation: Center for Proteomics, TGen (Translational Genomics Research Institute), Phoenix, AZ 85004, USA.
authors.forename: Kristine
authors.initials: K
authors.lastname: Tsantilas
authors.affiliation: Center for Proteomics, TGen (Translational Genomics Research Institute), Phoenix, AZ 85004, USA.
authors.forename: Krystine
authors.initials: K
authors.lastname: Garcia
authors.affiliation: Center for Proteomics, TGen (Translational Genomics Research Institute), Phoenix, AZ 85004, USA.
authors.forename: Patrick
authors.initials: P
authors.lastname: Pirrotte
authors.affiliation: Divisions of Neurology and Neurobiology, Barrow Neurological Institute, St. Joseph׳s Hospital and Medical Center, Phoenix, AZ 85013, USA; University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA. Electronic address: Robert.Bowser@DignityHealth.org.
authors.forename: Robert
authors.initials: R
authors.lastname: Bowser
mesh_terms: D000690:Amyotrophic Lateral Sclerosis / Q000378:metabolism*; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D057174:Frontotemporal Lobar Degeneration / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D002479:Inclusion Bodies / Q000378:metabolism; D013058:Mass Spectrometry; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010448:Peptide Initiation Factors / Q000378:metabolism; D060066:Protein Interaction Maps; D012326:RNA Splicing; D016601:RNA-Binding Proteins / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D015852:Eukaryotic Initiation Factor-2; D009419:Nerve Tissue Proteins; D010448:Peptide Initiation Factors; C467044:RBM45 protein, human; D016601:RNA-Binding Proteins; C075833:eIF-4
keywords: ALS; Immunoprecipitation; Mass spectrometry; Protein–protein interaction; RBM45