title: Detection of Protein-Protein Interaction Within an RNA-Protein Complex Via Unnatural-Amino-Acid-Mediated Photochemical Crosslinking.
abstract: Although DExD/H-box proteins are known to unwind RNA duplexes and modulate RNA structures in vitro, it is highly plausible that, in vivo, some may function to remodel RNA-protein complexes. Precisely how the latter is achieved remains a mystery. We investigated this critical issue by using yeast Prp28p, an evolutionarily conserved DExD/H-box splicing factor, as a model system. To probe how Prp28p interacts with spliceosome, we strategically placed p-benzoyl-phenylalanine (BPA), a photoactivatable unnatural amino acid, along the body of Prp28p in vivo. Extracts prepared from these engineered strains were then used to assemble in vitro splicing reactions for BPA-mediated protein-protein crosslinkings. This enabled us, for the first time, to "capture" Prp28p in action. This approach may be applicable to studying the roles of other DExD/H-box proteins functioning in diverse RNA-related pathways, as well as to investigating protein-protein contacts within an RNA-protein complex.
authors.affiliation: Genomics Research Center, Academia Sinica, 128 Academia Road, Section 2, Nankang, Taipei, 115, Taiwan.
authors.forename: Fu-Lung
authors.initials: FL
authors.lastname: Yeh
authors.affiliation: Genomics Research Center, Academia Sinica, 128 Academia Road, Section 2, Nankang, Taipei, 115, Taiwan.
authors.forename: Luh
authors.initials: L
authors.lastname: Tung
authors.affiliation: Genomics Research Center, Academia Sinica, 128 Academia Road, Section 2, Nankang, Taipei, 115, Taiwan. chang108@gate.sinica.edu.tw.
authors.forename: Tien-Hsien
authors.initials: TH
authors.lastname: Chang
mesh_terms: D053487:DEAD-box RNA Helicases / Q000378:metabolism*; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000378:metabolism*; D055668:Photochemical Processes; D025941:Protein Interaction Mapping / Q000379:methods*; D060066:Protein Interaction Maps; D012326:RNA Splicing; D012331:RNA, Fungal / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D017381:Spliceosomes / Q000378:metabolism*
chemical_list: D012331:RNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D010649:Phenylalanine; C088939:PRP28 protein, S cerevisiae; D053487:DEAD-box RNA Helicases
keywords: BPA; DExD/H-box protein; Pre-mRNA splicing; Prp28p; Prp8p; RNA helicase; RNA-dependent ATPase; RNPase; UV crosslinking; p-benzoyl-phenylalanine