title: Structure/Function Analysis of Protein-Protein Interactions Developed by the Yeast Pih1 Platform Protein and Its Partners in Box C/D snoRNP Assembly.
abstract: In eukaryotes, nucleotide post-transcriptional modifications in RNAs play an essential role in cell proliferation by contributing to pre-ribosomal RNA processing, ribosome assembly and activity. Box C/D small nucleolar ribonucleoparticles catalyze site-specific 2'-O-methylation of riboses, one of the most prevalent RNA modifications. They contain one guide RNA and four core proteins and their in vivo assembly requires numerous factors including (HUMAN/Yeast) BCD1/Bcd1p, NUFIP1/Rsa1p, ZNHIT3/Hit1p, the R2TP complex composed of protein PIH1D1/Pih1p and RPAP3/Tah1p that bridges the R2TP complex to the HSP90/Hsp82 chaperone and two AAA+ ATPases. We show that Tah1p can stabilize Pih1p in the absence of Hsp82 activity during the stationary phase of growth and consequently that the Tah1p:Pih1p interaction is sufficient for Pih1p stability. This prompted us to establish the solution structure of the Tah1p:Pih1p complex by NMR. The C-terminal tail S93-S111 of Tah1p snakes along Pih1p264-344 folded in a CS domain to form two intermolecular β-sheets and one covering loop. However, a thorough inspection of the NMR and crystal structures revealed structural differences that may be of functional importance. In addition, our NMR and isothermal titration calorimetry data revealed the formation of direct contacts between Pih1p257-344 and the Hsp82MC domain in the presence of Tah1p. By co-expression in Escherichia coli, we demonstrate that Pih1p has two other direct partners, the Rsa1p assembly factor and the Nop58p core protein, and in vivo and in vitro experiments mapped the required binding domains. Our data suggest that these two interactions are mutually exclusive. The implication of this finding for box C/D small nucleolar ribonucleoparticle assembly is discussed.
authors.affiliation: FR 3209 CNRS-Université de Lorraine, Bioingénierie Moléculaire, Cellulaire et Thérapeutique, Biopôle, Campus Biologie-Santé, CS 50184, 54505 Vandœuvre-lès-Nancy Cedex, France.
authors.forename: Marc
authors.initials: M
authors.lastname: Quinternet
authors.affiliation: Ingénierie Moléculaire et Physiopathologie Articulaire, UMR 7365 CNRS-Université de Lorraine, Biopôle, Campus Biologie Santé, 9 Avenue de la Forêt de Haye, CS 50184, 54505 Vandœuvre-lès-Nancy Cedex, France.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Rothé
authors.affiliation: Ingénierie Moléculaire et Physiopathologie Articulaire, UMR 7365 CNRS-Université de Lorraine, Biopôle, Campus Biologie Santé, 9 Avenue de la Forêt de Haye, CS 50184, 54505 Vandœuvre-lès-Nancy Cedex, France.
authors.forename: Muriel
authors.initials: M
authors.lastname: Barbier
authors.affiliation: Ingénierie Moléculaire et Physiopathologie Articulaire, UMR 7365 CNRS-Université de Lorraine, Biopôle, Campus Biologie Santé, 9 Avenue de la Forêt de Haye, CS 50184, 54505 Vandœuvre-lès-Nancy Cedex, France.
authors.forename: Claude
authors.initials: C
authors.lastname: Bobo
authors.affiliation: BioOrganic Mass Spectrometry Laboratory, IPHC-DSA, UMR 7178 Université de Strasbourg-CNRS, 25 rue Becquerel, 67087 Strasbourg, France.
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Saliou
authors.affiliation: Ingénierie Moléculaire et Physiopathologie Articulaire, UMR 7365 CNRS-Université de Lorraine, Biopôle, Campus Biologie Santé, 9 Avenue de la Forêt de Haye, CS 50184, 54505 Vandœuvre-lès-Nancy Cedex, France.
authors.forename: Clémence
authors.initials: C
authors.lastname: Jacquemin
authors.affiliation: Ingénierie Moléculaire et Physiopathologie Articulaire, UMR 7365 CNRS-Université de Lorraine, Biopôle, Campus Biologie Santé, 9 Avenue de la Forêt de Haye, CS 50184, 54505 Vandœuvre-lès-Nancy Cedex, France.
authors.forename: Régis
authors.initials: R
authors.lastname: Back
authors.affiliation: Ingénierie Moléculaire et Physiopathologie Articulaire, UMR 7365 CNRS-Université de Lorraine, Biopôle, Campus Biologie Santé, 9 Avenue de la Forêt de Haye, CS 50184, 54505 Vandœuvre-lès-Nancy Cedex, France.
authors.forename: Marie-Eve
authors.initials: ME
authors.lastname: Chagot
authors.affiliation: BioOrganic Mass Spectrometry Laboratory, IPHC-DSA, UMR 7178 Université de Strasbourg-CNRS, 25 rue Becquerel, 67087 Strasbourg, France.
authors.forename: Sarah
authors.initials: S
authors.lastname: Cianférani
authors.affiliation: Sorbonne Universités, UPMC University Paris 6, CNRS, UMR 8226, Laboratoire de Biologie Moléculaire et Cellulaire des Eucaryotes, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Meyer
authors.affiliation: Ingénierie Moléculaire et Physiopathologie Articulaire, UMR 7365 CNRS-Université de Lorraine, Biopôle, Campus Biologie Santé, 9 Avenue de la Forêt de Haye, CS 50184, 54505 Vandœuvre-lès-Nancy Cedex, France.
authors.forename: Christiane
authors.initials: C
authors.lastname: Branlant
authors.affiliation: Ingénierie Moléculaire et Physiopathologie Articulaire, UMR 7365 CNRS-Université de Lorraine, Biopôle, Campus Biologie Santé, 9 Avenue de la Forêt de Haye, CS 50184, 54505 Vandœuvre-lès-Nancy Cedex, France.
authors.forename: Bruno
authors.initials: B
authors.lastname: Charpentier
authors.affiliation: Ingénierie Moléculaire et Physiopathologie Articulaire, UMR 7365 CNRS-Université de Lorraine, Biopôle, Campus Biologie Santé, 9 Avenue de la Forêt de Haye, CS 50184, 54505 Vandœuvre-lès-Nancy Cedex, France. Electronic address: xavier.manival@univ-lorraine.fr.
authors.forename: Xavier
authors.initials: X
authors.lastname: Manival
mesh_terms: D004926:Escherichia coli / Q000235:genetics; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D008745:Methylation; D018832:Molecular Chaperones / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D009687:Nuclear Proteins / Q000378:metabolism*; D011485:Protein Binding; D017510:Protein Folding; D060066:Protein Interaction Maps; D017434:Protein Structure, Tertiary; D012335:RNA, Ribosomal / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear / Q000235:genetics / Q000378:metabolism*; D020718:Ribonucleoproteins, Small Nucleolar / Q000378:metabolism; D012269:Ribosomal Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C074510:HSP82 protein, S cerevisiae; D018841:HSP90 Heat-Shock Proteins; D018832:Molecular Chaperones; C438199:NOP58 protein, S cerevisiae; D009687:Nuclear Proteins; C497702:PIH1 protein, S cerevisiae; D012335:RNA, Ribosomal; C402485:RSA1 protein, S cerevisiae; D017411:Ribonucleoproteins, Small Nuclear; D020718:Ribonucleoproteins, Small Nucleolar; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins; C571197:Tah1 protein, S cerevisiae
keywords: HSP90/Hsp82; NMR; Pih1p:Nop58p interaction; RPAP3/Tah1p:PIH1D1/Pih1p complex; U3 snoRNA