title: Targeted cross-linking-mass spectrometry determines vicinal interactomes within heterogeneous RNP complexes.
abstract: Proteomic and RNomic approaches have identified many components of different ribonucleoprotein particles (RNPs), yet still little is known about the organization and protein proximities within these heterogeneous and highly dynamic complexes. Here we describe a targeted cross-linking approach, which combines cross-linking from a known anchor site with affinity purification and mass spectrometry (MS) to identify the changing vicinity interactomes along RNP maturation pathways. Our method confines the reaction radius of a heterobifunctional cross-linker to a specific interaction surface, increasing the probability to capture low abundance conformations and transient vicinal interactors too infrequent for identification by traditional cross-linking-MS approaches, and determine protein proximities within RNPs. Applying the method to two conserved RNA-associated complexes in Saccharomyces cerevisae, the mRNA export receptor Mex67:Mtr2 and the pre-ribosomal Nop7 subcomplex, we identified dynamic vicinal interactomes within those complexes and along their changing pathway milieu. Our results therefore show that this method provides a new tool to study the changing spatial organization of heterogeneous dynamic RNP complexes.
authors.affiliation: Department for Systems Biology, Institut de recherches cliniques de Montréal, Montréal, Québec H2W 1R7, Canada Département de biochimie, Faculté de médecine, Université de Montréal, Montréal, Québec H3T 1J4, Canada.
authors.forename: Christian
authors.initials: C
authors.lastname: Trahan
authors.affiliation: Department for Systems Biology, Institut de recherches cliniques de Montréal, Montréal, Québec H2W 1R7, Canada Département de biochimie, Faculté de médecine, Université de Montréal, Montréal, Québec H3T 1J4, Canada Division of Experimental Medicine, Faculty of Medicine, McGill University, Montréal, Québec H3A 1A3, Canada marlene.oeffinger@ircm.qc.ca.
authors.forename: Marlene
authors.initials: M
authors.lastname: Oeffinger
mesh_terms: D003432:Cross-Linking Reagents / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism; D029543:Nucleocytoplasmic Transport Proteins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods; D060066:Protein Interaction Maps; D017434:Protein Structure, Tertiary; D020543:Proteome / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D003432:Cross-Linking Reagents; D034441:Heterogeneous-Nuclear Ribonucleoproteins; C106898:MEX67 protein, S cerevisiae; D026901:Membrane Transport Proteins; C480169:Mtr2 protein, S cerevisiae; D046912:Multiprotein Complexes; C451799:NOP7 protein, S cerevisiae; D009687:Nuclear Proteins; D029543:Nucleocytoplasmic Transport Proteins; D020543:Proteome; D016601:RNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins