Immunoprecipitation and mass spectrometry defines an extensive RBM45 protein–protein interaction network

Yang Li, Mahlon Collins, Jiyan An, Rachel Geiser, Tony Tegeler, Kristine Tsantilas, Krystine Garcia, Patrick Pirrotte, Robert Bowser

Research output: Contribution to journalReview article

19 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)79-93
Number of pages15
JournalBrain Research
Volume1647
DOIs
StatePublished - Sep 15 2016

Keywords

  • ALS
  • Immunoprecipitation
  • Mass spectrometry
  • Protein–protein interaction
  • RBM45

ASJC Scopus subject areas

  • Neuroscience(all)
  • Molecular Biology
  • Clinical Neurology
  • Developmental Biology

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    Li, Y., Collins, M., An, J., Geiser, R., Tegeler, T., Tsantilas, K., Garcia, K., Pirrotte, P., & Bowser, R. (2016). Immunoprecipitation and mass spectrometry defines an extensive RBM45 protein–protein interaction network. Brain Research, 1647, 79-93. https://doi.org/10.1016/j.brainres.2016.02.047