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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...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Cold Spring Harbor Laboratory Press
2016
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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. |
format | Online Article Text |
id | pubmed-4986900 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | Cold Spring Harbor Laboratory Press |
record_format | MEDLINE/PubMed |
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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