Journal
RNA
Volume 22, Issue 9, Pages 1467-1475Publisher
COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT
DOI: 10.1261/rna.057760.116
Keywords
RNA exosome; protein complex purification; ribonuclease
Categories
Funding
- National Institutes of Health [P41GM109824, P41GM103314, P50GM107632]
- Danish Council for Independent Research Technology and Production Sciences
- Danish National Research Foundation [DNRF58]
- Simons Foundation [349247]
- NIH [S10 OD019994-01, S10 RR029300-01, S10 RR017291-01, C06 RR017528-01-CEM]
- Agouron Institute [F00316]
- NYSTAR
- Novo Nordisk Fonden [NNF15OC0017010] Funding Source: researchfish
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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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