title: Purification of RNA-Protein Splicing Complexes Using a Tagged Protein from In Vitro Splicing Reaction Mixture.
abstract: In eukaryotes, pre-mRNA splicing is an essential step for gene expression. Splicing reactions have been well investigated by using in vitro splicing reactions with extracts prepared from cultured cells. Here, we describe protocols for the preparation of splicing-competent extracts from cells expressing a tagged spliceosomal protein. The whole-cell extracts are able to splice exogenously added pre-mRNA and the RNA-protein complex formed in the in vitro splicing reaction can be purified by immunoprecipitation using antibodies against the peptide tag on the splicing protein. The method described here to prepare splicing-active extracts from whole cells is particularly useful when studying pre-mRNA splicing in various cell types, and the expression of a tagged spliceosomal protein allows one to purify and analyze the RNA-protein complexes by simple immunoprecipitation.
authors.affiliation: Medical Innovation Center, Laboratory for Malignancy Control Research, Kyoto University Graduate School of Medicine, 53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto, 606-8507, Japan. kataoka.naoyuki.6m@kyoto-u.ac.jp.
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Kataoka
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D019154:Protein Splicing; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012313:RNA / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012322:RNA Precursors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012326:RNA Splicing; D013010:Sonication / Q000379:methods; D017381:Spliceosomes / Q000235:genetics / Q000378:metabolism
chemical_list: D011506:Proteins; D012322:RNA Precursors; D012313:RNA
keywords: Anti-Flag antibody; Immunoprecipitation; Sonication; Splicing; Whole-cell extracts