Purification and analysis of endogenous human RNA exosome complexes.
Purification and analysis of endogenous human RNA exosome complexes.
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DOI：
10.1261/rna.057760.116
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发表时间：
2016-09
期刊：
影响因子：
--
通讯作者：
LaCava J
中科院分区：
文献类型：
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作者：
Domanski M;Upla P;Rice WJ;Molloy KR;Ketaren NE;Stokes DL;Jensen TH;Rout MP;LaCava J
由于其在关键的RNA代谢过程中的重要性，核糖核溶解RNA外泌体复合物已经成为近二十年来深入研究的焦点。外泌体亚基组装、辅因子和底物相互作用、酶催化和结构的研究主要是使用酵母酿酒酵母或细菌中产生的复合物进行的。在这里，我们研究了来自人胚肾（HEK）293细胞的内源性外泌体的不同群体，并测试了它们的酶活性和结构完整性。我们描述了以适合体外生物化学和负染色透射电子显微镜的产率和纯度制备含有EXOSC 10的具有酶活性的内源性人外泌体的方法。这为设计用于测试推定的辅因子对人外泌体活性的体外作用的测定打开了大门，并将使内源性来源的制剂的结构研究成为可能。
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.