title: Proteomic and 3D structure analyses highlight the C/D box snoRNP assembly mechanism and its control.
abstract: In vitro, assembly of box C/D small nucleolar ribonucleoproteins (snoRNPs) involves the sequential recruitment of core proteins to snoRNAs. In vivo, however, assembly factors are required (NUFIP, BCD1, and the HSP90-R2TP complex), and it is unknown whether a similar sequential scheme applies. In this paper, we describe systematic quantitative stable isotope labeling by amino acids in cell culture proteomic experiments and the crystal structure of the core protein Snu13p/15.5K bound to a fragment of the assembly factor Rsa1p/NUFIP. This revealed several unexpected features: (a) the existence of a protein-only pre-snoRNP complex containing five assembly factors and two core proteins, 15.5K and Nop58; (b) the characterization of ZNHIT3, which is present in the protein-only complex but gets released upon binding to C/D snoRNAs; (c) the dynamics of the R2TP complex, which appears to load/unload RuvBL AAA(+) adenosine triphosphatase from pre-snoRNPs; and (d) a potential mechanism for preventing premature activation of snoRNP catalytic activity. These data provide a framework for understanding the assembly of box C/D snoRNPs.
authors.affiliation: Equipe labellisée Ligue contre le Cancer, Centre National de la Recherche Scientifique Unité Mixte de Recherche 5535, Institut de Génétique Moléculaire de Montpellier, 34293 Montpellier, Cedex 5, France.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Bizarro
authors.affiliation: Ingénierie Moléculaire et Physiopathologie Articulaire, Centre National de la Recherche Scientifique Unité Mixte de Recherche 7365, Université de Lorraine, Biopôle de l'Université de Lorraine, 54505 Vandoeuvre-les-Nancy Cedex, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Charron
authors.affiliation: Centre de Recherches de Biochimie Macromoléculaire, Unité Mixte de Recherche 5237, 34293 Montpellier, Cedex 5, France.
authors.forename: Séverine
authors.initials: S
authors.lastname: Boulon
authors.affiliation: Centre for Gene Regulation and Expression, University of Dundee, Dundee DD1 5EH, Scotland, UK.
authors.forename: Belinda
authors.initials: B
authors.lastname: Westman
authors.affiliation: Equipe labellisée Ligue contre le Cancer, Centre National de la Recherche Scientifique Unité Mixte de Recherche 5535, Institut de Génétique Moléculaire de Montpellier, 34293 Montpellier, Cedex 5, France.
authors.forename: Bérengère
authors.initials: B
authors.lastname: Pradet-Balade
authors.affiliation: Centre National de la Recherche Scientifique Unité Mixte de Recherche 5203, Institut de Génomique Fonctionnelle, F-34000 Montpellier, France Institut National de la Santé et de la Recherche Médicale, U661, F-34000 Montpellier, France Unité Mixte de Recherche 5203, Université de Montpellier 1 and Université de Montpellier 2, F-34000 Montpellier, France.
authors.forename: Franck
authors.initials: F
authors.lastname: Vandermoere
authors.affiliation: Ingénierie Moléculaire et Physiopathologie Articulaire, Centre National de la Recherche Scientifique Unité Mixte de Recherche 7365, Université de Lorraine, Biopôle de l'Université de Lorraine, 54505 Vandoeuvre-les-Nancy Cedex, France.
authors.forename: Marie-Eve
authors.initials: ME
authors.lastname: Chagot
authors.affiliation: Equipe labellisée Ligue contre le Cancer, Centre National de la Recherche Scientifique Unité Mixte de Recherche 5535, Institut de Génétique Moléculaire de Montpellier, 34293 Montpellier, Cedex 5, France.
authors.forename: Marie
authors.initials: M
authors.lastname: Hallais
authors.affiliation: Centre for Gene Regulation and Expression, University of Dundee, Dundee DD1 5EH, Scotland, UK.
authors.forename: Yasmeen
authors.initials: Y
authors.lastname: Ahmad
authors.affiliation: Munich Center for Integrated Protein Science (CiPS) and Department of Biology, Ludwig Maximilians University Munich, 82152 Planegg-Martinsried, Germany Munich Center for Integrated Protein Science (CiPS) and Department of Biology, Ludwig Maximilians University Munich, 82152 Planegg-Martinsried, Germany.
authors.forename: Heinrich
authors.initials: H
authors.lastname: Leonhardt
authors.affiliation: Centre for Gene Regulation and Expression, University of Dundee, Dundee DD1 5EH, Scotland, UK.
authors.forename: Angus
authors.initials: A
authors.lastname: Lamond
authors.affiliation: Ingénierie Moléculaire et Physiopathologie Articulaire, Centre National de la Recherche Scientifique Unité Mixte de Recherche 7365, Université de Lorraine, Biopôle de l'Université de Lorraine, 54505 Vandoeuvre-les-Nancy Cedex, France.
authors.forename: Xavier
authors.initials: X
authors.lastname: Manival
authors.affiliation: Ingénierie Moléculaire et Physiopathologie Articulaire, Centre National de la Recherche Scientifique Unité Mixte de Recherche 7365, Université de Lorraine, Biopôle de l'Université de Lorraine, 54505 Vandoeuvre-les-Nancy Cedex, France.
authors.forename: Christiane
authors.initials: C
authors.lastname: Branlant
authors.affiliation: Ingénierie Moléculaire et Physiopathologie Articulaire, Centre National de la Recherche Scientifique Unité Mixte de Recherche 7365, Université de Lorraine, Biopôle de l'Université de Lorraine, 54505 Vandoeuvre-les-Nancy Cedex, France.
authors.forename: Bruno
authors.initials: B
authors.lastname: Charpentier
authors.affiliation: Equipe labellisée Ligue contre le Cancer, Centre National de la Recherche Scientifique Unité Mixte de Recherche 5535, Institut de Génétique Moléculaire de Montpellier, 34293 Montpellier, Cedex 5, France celine.verheggen@igmm.cnrs.fr edouard.bertrand@igmm.cnrs.fr.
authors.forename: Céline
authors.initials: C
authors.lastname: Verheggen
authors.affiliation: Equipe labellisée Ligue contre le Cancer, Centre National de la Recherche Scientifique Unité Mixte de Recherche 5535, Institut de Génétique Moléculaire de Montpellier, 34293 Montpellier, Cedex 5, France celine.verheggen@igmm.cnrs.fr edouard.bertrand@igmm.cnrs.fr.
authors.forename: Edouard
authors.initials: E
authors.lastname: Bertrand
mesh_terms: D000074183:ATPases Associated with Diverse Cellular Activities; D000595:Amino Acid Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D018360:Crystallography, X-Ray; D004265:DNA Helicases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D000075723:Kruppel-Like Factor 6; D051741:Kruppel-Like Transcription Factors / Q000378:metabolism; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D040901:Proteomics / Q000379:methods; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear / Q000737:chemistry* / Q000378:metabolism; D020718:Ribonucleoproteins, Small Nucleolar / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D016415:Sequence Alignment; D014157:Transcription Factors
chemical_list: D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D018841:HSP90 Heat-Shock Proteins; C105465:KLF6 protein, human; D000075723:Kruppel-Like Factor 6; D051741:Kruppel-Like Transcription Factors; C403092:NUFIP1 protein, human; C477652:NUFIP2 protein, human; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D016601:RNA-Binding Proteins; D017411:Ribonucleoproteins, Small Nuclear; D020718:Ribonucleoproteins, Small Nucleolar; C402291:Snu13 protein, human; D014157:Transcription Factors; C403666:ZNF331 protein, human; C546329:ZNHIT3 protein, human; D012260:Ribonucleases; D000074183:ATPases Associated with Diverse Cellular Activities; D004265:DNA Helicases; C486549:RUVBL1 protein, human; C122079:RUVBL2 protein, human