title: Purification of native Argonaute complexes from the fission yeast Schizosaccharomyces pombe.
abstract: 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.
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA, USA.
authors.forename: Shane M
authors.initials: SM
authors.lastname: Buker
authors.forename: Mohammad R
authors.initials: MR
authors.lastname: Motamedi
mesh_terms: D060565:Argonaute Proteins; D001709:Biotechnology / Q000379:methods*; D015153:Blotting, Western; D002424:Caustics / Q000494:pharmacology; D011232:Chemical Precipitation / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D016133:Polymerase Chain Reaction; D016601:RNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D034743:RNA-Induced Silencing Complex / Q000302:isolation & purification* / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000254:growth & development / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D016622:Silver Staining; D014170:Transformation, Genetic; D014238:Trichloroacetic Acid / Q000494:pharmacology
chemical_list: C481643:Ago1 protein, S pombe; D060565:Argonaute Proteins; D002424:Caustics; D016601:RNA-Binding Proteins; D034743:RNA-Induced Silencing Complex; D029702:Schizosaccharomyces pombe Proteins; D014238:Trichloroacetic Acid