title: Studying RNA-Protein Interactions of Pre-mRNA Complexes by Mass Spectrometry.
abstract: RNA-protein interactions play a crucial role in gene expression. These interactions take place in so-called ribonucleoprotein (RNP) complexes. To investigate which proteins interact with RNA in these complexes, and how they do so, UV-light-induced cross-linking has proven to be a valuable, yet straightforward technique. UV irradiation induces a covalent bond between the RNA and the proteins, whereafter cross-linked proteins can be identified by mass spectrometric (MS) approaches. Moreover, the cross-linked region of the protein, and often the actual cross-linked amino acid, can be identified by state-of-the-art MS, as can the cross-linked RNA moiety. This protocol describes in detail how to isolate peptide-RNA oligonucleotide cross-links from UV-irradiated human pre-mRNA RNPs and to perform the subsequent MS investigation of these peptide-RNA conjugates in combination with a dedicated computational analysis, in order to obtain sequence information about the cross-linked peptide and oligoribonucleotide. The described workflow can be applied to any RNP, irrespective of its origin, e.g., RNPs assembled in vitro (as described here) or RNPs isolated from UV-irradiated cells, either ex vivo or in vivo.
authors.affiliation: Bioanalytical Mass Spectrometry Group, Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany; Bioanalytics Research Group, Institute for Clinical Chemistry, University Medical Center, Göttingen, Germany.
authors.forename: Saadia
authors.initials: S
authors.lastname: Qamar
authors.affiliation: Bioanalytical Mass Spectrometry Group, Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany; Bioanalytics Research Group, Institute for Clinical Chemistry, University Medical Center, Göttingen, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Kramer
authors.affiliation: Bioanalytical Mass Spectrometry Group, Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany; Bioanalytics Research Group, Institute for Clinical Chemistry, University Medical Center, Göttingen, Germany. Electronic address: henning.urlaub@mpibpc.mpg.de.
authors.forename: Henning
authors.initials: H
authors.lastname: Urlaub
mesh_terms: D001665:Binding Sites; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008958:Models, Molecular; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D012322:RNA Precursors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000068103:Serine-Arginine Splicing Factors; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014158:Transcription, Genetic; D014466:Ultraviolet Rays
chemical_list: D009687:Nuclear Proteins; D009843:Oligoribonucleotides; D010750:Phosphoproteins; D012322:RNA Precursors; D012261:Ribonucleoproteins; C000595776:SRSF8 protein, human; C073288:SRSF2 protein, human; D000068103:Serine-Arginine Splicing Factors; C070852:RNA polymerase SP6; D012321:DNA-Directed RNA Polymerases
keywords: Mass spectrometry; Protein; RNA; RNA–protein interactions; RNP; UV-induced cross-linking