Buker Shane M, Motamedi Mohammad R
Department of Cell Biology, Harvard Medical School, Boston, MA, USA.
Methods Mol Biol. 2011;725:1-13. doi: 10.1007/978-1-61779-046-1_1.
Small interfering (si) RNAs, produced by the RNA interference (RNAi)-mediated processing of long double-stranded (ds) RNAs, can inhibit gene expression by post-transcriptional or transcriptional gene silencing mechanisms. At the heart of all small RNA-mediated silencing lies the key RNAi effector protein Argonaute, which once loaded with small RNAs can recognize its target transcript by siRNA-RNA Watson-Crick base pairing interactions. In the fission yeast Schizosaccharomyces pombe, the formation of the epigenetically heritable centromeric heterochromatin requires RNAi proteins including the sole fission yeast Argonaute homolog, Ago1. Two distinct native Ago1 complexes have been purified and studied extensively, both of which are required for siRNA production and heterochromatin formation at the fission yeast centromeres. The purification and analysis of the Argonaute siRNA chaperone (ARC) complex and RNA-induced transcriptional silencing (RITS) complex have provided insight into the mechanism of siRNA-Ago1 loading and the cis recruitment of silencing complexes at fission yeast centromeres, respectively. These discoveries have been instrumental in shaping the current models of RNA-mediated epigenetic silencing in eukaryotes. Below, we describe the protocol used for affinity purification of the native Ago1 complexes from S. pombe.
小干扰（si）RNA由RNA干扰（RNAi）介导的长双链（ds）RNA加工产生，可通过转录后或转录基因沉默机制抑制基因表达。所有小RNA介导的沉默的核心是关键的RNAi效应蛋白AGO，一旦加载小RNA，AGO就能通过siRNA-RNA沃森-克里克碱基配对相互作用识别其靶转录本。在裂殖酵母粟酒裂殖酵母中，表观遗传可遗传的着丝粒异染色质的形成需要RNAi蛋白，包括唯一的裂殖酵母AGO同源物AGO1。两种不同天然AGO1复合物已被纯化并广泛研究，二者都是裂殖酵母着丝粒处siRNA产生和异染色质形成所必需的。AGO小RNA伴侣（ARC）复合物和RNA诱导转录沉默（RITS）复合物的纯化与分析，分别为裂殖酵母着丝粒处siRNA-AGO1加载机制和沉默复合物的顺式募集提供了见解。这些发现有助于构建当前真核生物中RNA介导的表观遗传沉默模型。下面，我们描述了从粟酒裂殖酵母中亲和纯化天然AGO1复合物的方法。
