title: In vitro RNA-binding assay for studying trans-factors for RNA editing in chloroplasts.
abstract: In plant organelles, specific C residues are modified to U by RNA editing. Short RNA sequences surrounding the target site (i.e., cis-elements) are recognized by trans-factors, which were recently shown to be pentatricopeptide repeat (PPR) proteins. PPR proteins consist of tandem arrays of a highly degenerate unit of 35 (pentatrico) amino acids, and PPR motifs are believed to recognize specific RNA sequences. In Arabidopsis thaliana, more than 450 sites are edited in mitochondria and plastids, and a similar number of PPR proteins are encoded in the nuclear genome. To study how the tandem array of a PPR motif facilitates the recognition of RNA sequences, an efficient biochemical strategy is an in vitro binding assay of recombinant PPR proteins with target RNA. This analysis is especially powerful with a combination of in vivo analyses based on the phenotypes of mutants and transgenic plants. In this chapter, we describe methods for the expression of recombinant PPR proteins in Escherichia coli, preparation of probe RNAs, and RNA gel shift assays. These methods can also be utilized for other RNA-binding proteins.
authors.affiliation: Department of Botany, Graduate School of Science, Kyoto University, Kyoto, Japan. shikanai@pmg.bot.kyoto-u.ac.jp
authors.forename: Toshiharu
authors.initials: T
authors.lastname: Shikanai
authors.forename: Kenji
authors.initials: K
authors.lastname: Okuda
mesh_terms: D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000378:metabolism*; D001681:Biological Assay / Q000379:methods*; D002736:Chloroplasts / Q000187:drug effects / Q000235:genetics*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D005822:Genetic Vectors / Q000235:genetics; D008163:Luminescent Measurements; D008954:Models, Biological; D017393:RNA Editing / Q000187:drug effects / Q000235:genetics*; D015347:RNA Probes / Q000378:metabolism; D018747:RNA, Chloroplast / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification; D013698:Templates, Genetic; D015534:Trans-Activators / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D029681:Arabidopsis Proteins; D003432:Cross-Linking Reagents; D015347:RNA Probes; D018747:RNA, Chloroplast; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D015534:Trans-Activators