Purification and analysis of endogenous human RNA exosome complexes

TY - JOUR

T1 - Purification and analysis of endogenous human RNA exosome complexes

AU - Domanski, Michal

AU - Upla, Paula

AU - Rice, William J.

AU - Molloy, Kelly R.

AU - Ketaren, Natalia E.

AU - Stokes, David L.

AU - Jensen, Torben Heick

AU - Rout, Michael P.

AU - Lacava, John

PY - 2016/9/1

Y1 - 2016/9/1

N2 - 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.

AB - 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.

KW - Protein complex purification

KW - Ribonuclease

KW - RNA exosome

UR - http://www.scopus.com/inward/record.url?scp=84983247234&partnerID=8YFLogxK

U2 - 10.1261/rna.057760.116

DO - 10.1261/rna.057760.116

M3 - Journal article

C2 - 27402899

AN - SCOPUS:84983247234

SN - 1355-8382

VL - 22

SP - 1467

EP - 1475

JO - RNA

JF - RNA

IS - 9

ER -