title: RNA interactome capture in yeast.
abstract: RNA-binding proteins (RBPs) are key players in post-transcriptional regulation of gene expression in eukaryotic cells. To be able to unbiasedly identify RBPs in Saccharomyces cerevisiae, we developed a yeast RNA interactome capture protocol which employs RNA labeling, covalent UV crosslinking of RNA and proteins at 365nm wavelength (photoactivatable-ribonucleoside-enhanced crosslinking, PAR-CL) and finally purification of the protein-bound mRNA. The method can be easily implemented in common workflows and takes about 3days to complete. Next to a comprehensive explanation of the method, we focus on our findings about the choice of crosslinking in yeast and discuss the rationale of individual steps in the protocol.
authors.affiliation: European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117 Heidelberg, Germany; IRI for Life Sciences & Institut für Biologie, Humboldt-Universität zu Berlin, Philippstrasse 13, 10115 Berlin, Germany. Electronic address: benedikt.beckmann@iri-lifesciences.de.
authors.forename: Benedikt M
authors.initials: BM
authors.lastname: Beckmann
mesh_terms: D000906:Antibodies / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D047468:Immunoprecipitation / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods; D055668:Photochemical Processes; D011485:Protein Binding; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D017423:Sequence Analysis, RNA / Q000379:methods; D013194:Staining and Labeling / Q000379:methods*; D013462:Sulfur Radioisotopes; D013889:Thiouracil / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D059467:Transcriptome; D014466:Ultraviolet Rays
chemical_list: D000906:Antibodies; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D013462:Sulfur Radioisotopes; C114719:4-thiouracil; D013889:Thiouracil; D012260:Ribonucleases
keywords: RBP; RNA labeling; UV crosslinking; Yeast; mRNA