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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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Autores principales: Domanski, Michal, Upla, Paula, Rice, William J., Molloy, Kelly R., Ketaren, Natalia E., Stokes, David L., Jensen, Torben Heick, Rout, Michael P., LaCava, John
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory Press 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4986900/
https://www.ncbi.nlm.nih.gov/pubmed/27402899
http://dx.doi.org/10.1261/rna.057760.116
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author Domanski, Michal
Upla, Paula
Rice, William J.
Molloy, Kelly R.
Ketaren, Natalia E.
Stokes, David L.
Jensen, Torben Heick
Rout, Michael P.
LaCava, John
author_facet Domanski, Michal
Upla, Paula
Rice, William J.
Molloy, Kelly R.
Ketaren, Natalia E.
Stokes, David L.
Jensen, Torben Heick
Rout, Michael P.
LaCava, John
author_sort Domanski, Michal
collection PubMed
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.
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spelling pubmed-49869002016-09-01 Purification and analysis of endogenous human RNA exosome complexes Domanski, Michal Upla, Paula Rice, William J. Molloy, Kelly R. Ketaren, Natalia E. Stokes, David L. Jensen, Torben Heick Rout, Michael P. LaCava, John RNA Method 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. Cold Spring Harbor Laboratory Press 2016-09 /pmc/articles/PMC4986900/ /pubmed/27402899 http://dx.doi.org/10.1261/rna.057760.116 Text en © 2016 Domanski et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society http://creativecommons.org/licenses/by/4.0/ This article, published in RNA, is available under a Creative Commons License (Attribution 4.0 International), as described at http://creativecommons.org/licenses/by/4.0/.
spellingShingle Method
Domanski, Michal
Upla, Paula
Rice, William J.
Molloy, Kelly R.
Ketaren, Natalia E.
Stokes, David L.
Jensen, Torben Heick
Rout, Michael P.
LaCava, John
Purification and analysis of endogenous human RNA exosome complexes
title Purification and analysis of endogenous human RNA exosome complexes
title_full Purification and analysis of endogenous human RNA exosome complexes
title_fullStr Purification and analysis of endogenous human RNA exosome complexes
title_full_unstemmed Purification and analysis of endogenous human RNA exosome complexes
title_short Purification and analysis of endogenous human RNA exosome complexes
title_sort purification and analysis of endogenous human rna exosome complexes
topic Method
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4986900/
https://www.ncbi.nlm.nih.gov/pubmed/27402899
http://dx.doi.org/10.1261/rna.057760.116
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