Purification and analysis of endogenous human RNA exosome complexes

Michal Domanski; Paula Upla; William J Rice; Kelly R Molloy; Natalia E Ketaren; David L Stokes; Torben Heick Jensen; Michael P Rout; John LaCava

doi:10.1261/rna.057760.116

Purification and analysis of endogenous human RNA exosome complexes

RNA. 2016 Sep;22(9):1467-75. doi: 10.1261/rna.057760.116. Epub 2016 Jul 11.

Authors

Michal Domanski¹, Paula Upla², William J Rice³, Kelly R Molloy⁴, Natalia E Ketaren⁵, David L Stokes⁶, Torben Heick Jensen⁷, Michael P Rout⁵, John LaCava⁸

Affiliations

¹ 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.
² 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.
³ Simons Electron Microscopy Center at New York Structural Biology Center, New York, New York 10027, USA.
⁴ Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, New York 10065, USA.
⁵ Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, New York 10065, USA.
⁶ Skirball Institute and Department of Cell Biology, New York University School of Medicine, New York, New York 10016, USA.
⁷ Centre for mRNP Biogenesis and Metabolism, Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark.
⁸ 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.

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.

Keywords: RNA exosome; protein complex purification; ribonuclease.

Publication types

Research Support, Non-U.S. Gov't
Research Support, N.I.H., Extramural

MeSH terms

Exosomes / chemistry*
Exosomes / metabolism
HEK293 Cells
Humans
RNA, Messenger / chemistry
RNA, Messenger / metabolism

Substances

RNA, Messenger

Abstract

Publication types

MeSH terms

Substances

Grants and funding