title: Purification and analysis of endogenous human RNA exosome complexes.
abstract: As a result of its importance in key RNA metabolic processes, the ribonucleolytic RNA exosome complex has been the focus of intense study for almost two decades. Research on exosome subunit assembly, cofactor and substrate interaction, enzymatic catalysis and structure have largely been conducted using complexes produced in the yeast Saccharomyces cerevisiae or in bacteria. Here, we examine different populations of endogenous exosomes from human embryonic kidney (HEK) 293 cells and test their enzymatic activity and structural integrity. We describe methods to prepare EXOSC10-containing, enzymatically active endogenous human exosomes at suitable yield and purity for in vitro biochemistry and negative stain transmission electron microscopy. This opens the door for assays designed to test the in vitro effects of putative cofactors on human exosome activity and will enable structural studies of preparations from endogenous sources.
authors.affiliation: Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, New York 10065, USA Centre for mRNP Biogenesis and Metabolism, Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark.
authors.forename: Michal
authors.initials: M
authors.lastname: Domanski
authors.affiliation: Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, New York 10065, USA Skirball Institute and Department of Cell Biology, New York University School of Medicine, New York, New York 10016, USA.
authors.forename: Paula
authors.initials: P
authors.lastname: Upla
authors.affiliation: Simons Electron Microscopy Center at New York Structural Biology Center, New York, New York 10027, USA.
authors.forename: William J
authors.initials: WJ
authors.lastname: Rice
authors.affiliation: Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, New York 10065, USA.
authors.forename: Kelly R
authors.initials: KR
authors.lastname: Molloy
authors.affiliation: Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, New York 10065, USA.
authors.forename: Natalia E
authors.initials: NE
authors.lastname: Ketaren
authors.affiliation: Skirball Institute and Department of Cell Biology, New York University School of Medicine, New York, New York 10016, USA.
authors.forename: David L
authors.initials: DL
authors.lastname: Stokes
authors.affiliation: Centre for mRNP Biogenesis and Metabolism, Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark.
authors.forename: Torben Heick
authors.initials: TH
authors.lastname: Jensen
authors.affiliation: Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, New York 10065, USA.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Rout
authors.affiliation: Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, New York 10065, USA Institute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, New York 10016, USA.
authors.forename: John
authors.identifier: 0000-0002-6307-7713
authors.initials: J
authors.lastname: LaCava
mesh_terms: D055354:Exosomes / Q000737:chemistry* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism
chemical_list: D012333:RNA, Messenger
keywords: RNA exosome; protein complex purification; ribonuclease