Purification and analysis of endogenous human RNA exosome complexes

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 us...

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Bibliographic Details
Published in:RNA (Cambridge) 2016-09, Vol.22 (9), p.1467-1475
Main Authors: Domanski, Michal, Upla, Paula, Rice, William J, Molloy, Kelly R, Ketaren, Natalia E, Stokes, David L, Jensen, Torben Heick, Rout, Michael P, LaCava, John
Format: Article
Language:English
Subjects:
Exosomes - chemistry
Exosomes - metabolism
HEK293 Cells
Humans
Method
RNA, Messenger - chemistry
RNA, Messenger - metabolism
Saccharomyces cerevisiae
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Summary: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.
ISSN:1355-8382
1469-9001
DOI:10.1261/rna.057760.116