title: Production of pure and functional RNA for in vitro reconstitution experiments.
abstract: Reconstitution of protein complexes has been a valuable tool to test molecular functions and to interpret in vivo observations. In recent years, a large number of RNA-protein complexes has been identified to regulate gene expression and to be important for a range of cellular functions. In contrast to protein complexes, in vitro analyses of RNA-protein complexes are hampered by the fact that recombinant expression and purification of RNA molecules is more difficult and less well established than for proteins. Here we review the current state of technology available for in vitro experiments with RNAs. We outline the possibilities to produce and purify large amounts of homogenous RNA and to perform the required quality controls. RNA-specific problems such as degradation, 5' and 3' end heterogeneity, co-existence of different folding states, and prerequisites for reconstituting RNAs with recombinantly expressed proteins are discussed. Additionally a number of techniques for the characterization of direct and indirect RNA-protein interactions are explained.
authors.affiliation: Institute of Structural Biology, Helmholtz Zentrum München, German Research Center for Environmental Health, Ingolstädter Landstraße 1, 85764 Neuherberg, Germany.
authors.forename: Franziska Theresia
authors.initials: FT
authors.lastname: Edelmann
authors.affiliation: Institute of Structural Biology, Helmholtz Zentrum München, German Research Center for Environmental Health, Ingolstädter Landstraße 1, 85764 Neuherberg, Germany.
authors.forename: Annika
authors.initials: A
authors.lastname: Niedner
authors.affiliation: Institute of Structural Biology, Helmholtz Zentrum München, German Research Center for Environmental Health, Ingolstädter Landstraße 1, 85764 Neuherberg, Germany. Electronic address: niessing@helmholtz-muenchen.de.
authors.forename: Dierk
authors.initials: D
authors.lastname: Niessing
mesh_terms: D008967:Molecular Biology / Q000379:methods*; D009690:Nucleic Acid Conformation; D020871:RNA Stability; D012343:RNA, Transfer / Q000096:biosynthesis* / Q000302:isolation & purification; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics; D014158:Transcription, Genetic
chemical_list: D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; C471175:SHE2 protein, S cerevisiae; C471176:SHE3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D012343:RNA, Transfer
keywords: RNA; RNA folding; RNA purification; RNA quality; RNA–protein interactions; RNP reconstitution