title: Analysis of protein-RNA interactions in CRISPR proteins and effector complexes by UV-induced cross-linking and mass spectrometry.
abstract: Ribonucleoprotein (RNP) complexes play important roles in the cell by mediating basic cellular processes, including gene expression and its regulation. Understanding the molecular details of these processes requires the identification and characterization of protein-RNA interactions. Over the years various approaches have been used to investigate these interactions, including computational analyses to look for RNA binding domains, gel-shift mobility assays on recombinant and mutant proteins as well as co-crystallization and NMR studies for structure elucidation. Here we report a more specialized and direct approach using UV-induced cross-linking coupled with mass spectrometry. This approach permits the identification of cross-linked peptides and RNA moieties and can also pin-point exact RNA contact sites within the protein. The power of this method is illustrated by the application to different single- and multi-subunit RNP complexes belonging to the prokaryotic adaptive immune system, CRISPR-Cas (CRISPR: clustered regularly interspaced short palindromic repeats; Cas: CRISPR associated). In particular, we identified the RNA-binding sites within three Cas7 protein homologs and mapped the cross-linking results to reveal structurally conserved Cas7 - RNA binding interfaces. These results demonstrate the strong potential of UV-induced cross-linking coupled with mass spectrometry analysis to identify RNA interaction sites on the RNA binding proteins.
authors.affiliation: Bioanalytical Mass Spectrometry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
authors.forename: Kundan
authors.initials: K
authors.lastname: Sharma
authors.affiliation: Structural Cell Biology Department, Max Planck Institute for Biochemistry, Martinsried, Germany.
authors.forename: Ajla
authors.initials: A
authors.lastname: Hrle
authors.affiliation: Bioanalytical Mass Spectrometry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany; Plant Proteomics Group, Max Planck Institute for Plant Breeding Research, Cologne, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Kramer
authors.affiliation: Center for Bioinformatics, University of Tübingen, Tübingen, Germany; Department of Computer Science, University of Tübingen, Tübingen, Germany.
authors.forename: Timo
authors.initials: T
authors.lastname: Sachsenberg
authors.affiliation: Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Wageningen, The Netherlands.
authors.forename: Raymond H J
authors.initials: RH
authors.lastname: Staals
authors.affiliation: Prokaryotic Small RNA Biology, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany.
authors.forename: Lennart
authors.initials: L
authors.lastname: Randau
authors.affiliation: Department of Biology II, Ulm University, Ulm, Germany.
authors.forename: Anita
authors.initials: A
authors.lastname: Marchfelder
authors.affiliation: Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Wageningen, The Netherlands.
authors.forename: John
authors.initials: J
authors.lastname: van der Oost
authors.affiliation: Center for Bioinformatics, University of Tübingen, Tübingen, Germany; Department of Computer Science, University of Tübingen, Tübingen, Germany; Quantitative Biology Center, University of Tübingen, Tübingen, Germany; Faculty of Medicine, University of Tübingen, Tübingen, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Kohlbacher
authors.affiliation: Structural Cell Biology Department, Max Planck Institute for Biochemistry, Martinsried, Germany.
authors.forename: Elena
authors.initials: E
authors.lastname: Conti
authors.affiliation: Bioanalytical Mass Spectrometry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany; Bioanalytics Research Group, Department of Clinical Chemistry, University Medical Center, Göttingen, Germany.
authors.forename: Henning
authors.initials: H
authors.lastname: Urlaub
mesh_terms: D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000502:physiology*; D010775:Photic Stimulation / Q000379:methods; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012313:RNA / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014466:Ultraviolet Rays
chemical_list: D016601:RNA-Binding Proteins; D012313:RNA
keywords: CRISPR-Cas; Cas7; Mass spectrometry; Protein–RNA interactions; UV cross-linking