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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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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cited_by cdi_FETCH-LOGICAL-c423t-d308c81ec76b1e5f9a472c1de608994b51d9a82149aec35010e526fc19b8ff843
cites cdi_FETCH-LOGICAL-c423t-d308c81ec76b1e5f9a472c1de608994b51d9a82149aec35010e526fc19b8ff843
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container_issue 9
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container_title RNA (Cambridge)
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creator Domanski, Michal
Upla, Paula
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description 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.
doi_str_mv 10.1261/rna.057760.116
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subjects Exosomes - chemistry
Exosomes - metabolism
HEK293 Cells
Humans
Method
RNA, Messenger - chemistry
RNA, Messenger - metabolism
Saccharomyces cerevisiae
title Purification and analysis of endogenous human RNA exosome complexes
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