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2. The box C/D snoRNP assembly factor Bcd1 interacts with the histone chaperone Rtt106 and controls its transcription dependent activity

Author

Bragantini, Benoît, Charron, Christophe, Bourguet, Maxime, Paul, Arnaud, Tiotiu, Decebal, Rothé, Benjamin, Marty, Hélène, Terral, Guillaume, Hessmann, Steve, Decourty, Laurence, Chagot, Marie-Eve, Strub, Jean-Marc, Massenet, Séverine, Bertrand, Edouard, Quinternet, Marc, Saveanu, Cosmin, Cianférani, Sarah, Labialle, Stéphane, Manival, Xavier, and Charpentier, Bruno

Published
2021
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6. Deciphering cellular and molecular determinants of human DPCD protein in complex with RUVBL1/RUVBL2 AAA-ATPases

Author

Dos Santos Morais, Raphael, primary, Santo, Paulo E., additional, Ley, Marie, additional, Schelcher, Cédric, additional, Abel, Yoann, additional, Plassart, Laura, additional, Deslignière, Evolène, additional, Chagot, Marie-Eve, additional, Quinternet, Marc, additional, Paiva, Ana C.F., additional, Hessmann, Steve, additional, Morellet, Nelly, additional, M. F. Sousa, Pedro, additional, Vandermoere, Franck, additional, Bertrand, Edouard, additional, Charpentier, Bruno, additional, Bandeiras, Tiago M., additional, Plisson-Chastang, Célia, additional, Verheggen, Céline, additional, Cianférani, Sarah, additional, and Manival, Xavier, additional

Published
2022
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9. The interaction between RPAP3 and TRBP reveals a possible involvement of the HSP90/R2TP chaperone complex in the regulation of miRNA activity

Author

Abel, Yoann, primary, Charron, Christophe, additional, Virciglio, Camille, additional, Bourguignon-Igel, Valérie, additional, Quinternet, Marc, additional, Chagot, Marie-Eve, additional, Robert, Marie-Cécile, additional, Verheggen, Céline, additional, Branlant, Christiane, additional, Bertrand, Edouard, additional, Manival, Xavier, additional, Charpentier, Bruno, additional, and Rederstorff, Mathieu, additional

Published
2022
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11. NOPCHAP1 is a PAQosome cofactor that helps loading NOP58 on RUVBL1/2 during box C/D snoRNP biogenesis

Author

Abel, Yoann, primary, Paiva, Ana C F, additional, Bizarro, Jonathan, additional, Chagot, Marie-Eve, additional, Santo, Paulo E, additional, Robert, Marie-Cécile, additional, Quinternet, Marc, additional, Vandermoere, Franck, additional, Sousa, Pedro M F, additional, Fort, Philippe, additional, Charpentier, Bruno, additional, Manival, Xavier, additional, Bandeiras, Tiago M, additional, Bertrand, Edouard, additional, and Verheggen, Céline, additional

Published
2020
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12. Structure of the RPAP3:TRBP interaction reveals an involvement of the HSP90/R2TP chaperone complex in dsRNA pathways

Author

Abel, Yoann, primary, Charron, Christophe, additional, Bourguignon-Igel, Valérie, additional, Quinternet, Marc, additional, Chagot, Marie-Eve, additional, Verheggen, Céline, additional, Branlant, Christiane, additional, Bertrand, Edouard, additional, Manival, Xavier, additional, Charpentier, Bruno, additional, and Rederstorff, Mathieu, additional

Published
2020
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15. The RPAP3-Cterminal domain identifies R2TP-like quaternary chaperones

Author

Maurizy, Chloe, Quinternet, Marc, Abel, Yoann, Verheggen, Celine, Santo, Paulo E., Bourguet, Maxime, Paiva, Ana C. F., Bragantini, Benoit, Chagot, Marie-Eve, Robert, Marie-Cecile, Abeza, Claire, Fabre, Philippe, Fort, Philippe, Vandermoere, Franck, Sousa, Pedro M. F., Rain, Jean-Christophe, Charpentier, Bruno, Cianferani, Sarah, Bandeiras, Tiago M., Pradet-Balade, Berengere, Manival, Xavier, Bertrand, Edouard, Institut de Génomique Fonctionnelle (IGF), Université de Montpellier (UM)-Université Montpellier 1 (UM1)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Montpellier 2 - Sciences et Techniques (UM2)-Centre National de la Recherche Scientifique (CNRS), Ingénierie, Biologie et Santé en Lorraine (IBSLor), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS), Ingénierie Moléculaire et Physiopathologie Articulaire (IMoPA), Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS), Institut de Génétique Moléculaire de Montpellier (IGMM), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Laboratoire de Spectrométrie de Masse BioOrganique (IPHC-DSA), Instituto de Biologia Experimental e Tecnológica (IBET), Centre de recherches de biochimie macromoléculaire (CRBM), Université Montpellier 1 (UM1)-Université Montpellier 2 - Sciences et Techniques (UM2)-IFR122-Centre National de la Recherche Scientifique (CNRS), Hybrigenics [Paris], Hybrigenics, Service de néphrologie, Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Hôpital Bicêtre-Institut National de la Santé et de la Recherche Médicale (INSERM), Instituto de Tecnologia Química e Biológica António Xavier (ITQB), Universidade Nova de Lisboa (NOVA), Equipe labellisée Ligue contre le Cancer, Université de Lorraine (UL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Universidade de Lisboa (ULISBOA), Laboratoire de Spectrométrie de Masse BioOrganique [Strasbourg] (LSMBO), Sciences Analytiques et Interactions Ioniques et Biomoléculaires (DSA-IPHC), Institut Pluridisciplinaire Hubert Curien (IPHC), Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Institut Pluridisciplinaire Hubert Curien (IPHC), Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS), Centre de recherche en Biologie Cellulaire (CRBM), Université Montpellier 1 (UM1)-Université Montpellier 2 - Sciences et Techniques (UM2)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Institut Pluridisciplinaire Hubert Curien (IPHC), Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Université de Montpellier (UM), Laboratoire de Spectrométrie de Masse BioOrganique, Département des Sciences Analytiques, Institut Pluridisciplinaire Hubert Curien, Strasbourg, France (LSMBO-DSA-IPHC), Centre National de la Recherche Scientifique (CNRS), Département Sciences Analytiques et Interactions Ioniques et Biomoléculaires (DSA-IPHC), Université Montpellier 2 - Sciences et Techniques (UM2)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Université Montpellier 1 (UM1), bertrand, edouard, Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Universidade de Lisboa = University of Lisbon (ULISBOA), Centre de recherche en Biologie cellulaire de Montpellier (CRBM), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Male, AAA proteins, [SDV]Life Sciences [q-bio], General Physics and Astronomy, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Chaperone, [SDV.BID.SPT]Life Sciences [q-bio]/Biodiversity/Systematics, Phylogenetics and taxonomy, Protein Structure, Secondary, Testis, lcsh:Science, GeneralLiterature_REFERENCE(e.g.,dictionaries,encyclopedias,glossaries), ComputingMilieux_MISCELLANEOUS, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], [SDV] Life Sciences [q-bio], [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Antigens, Surface, Protein Binding, Signal Transduction, Cell biology, RUVBL2, Science, [SDV.CAN]Life Sciences [q-bio]/Cancer, Hsp90, Chaperone (protein), [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Plasma protein binding, Article, General Biochemistry, Genetics and Molecular Biology, Cell Line, GTP-Binding Proteins, Genetics, Humans, GTP-binding protein regulators, HSP90 Heat-Shock Proteins, Protein folding, IP LUMIER, Adaptor Proteins, Signal Transducing, DNA Helicases, Membrane Proteins, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, General Chemistry, liprin-α2, NMR, [SDV.BDD.EO]Life Sciences [q-bio]/Development Biology/Embryology and Organogenesis, HEK293 Cells, Membrane protein, ATPases Associated with Diverse Cellular Activities, lcsh:Q, Apoptosis Regulatory Proteins, Carrier Proteins, R2SP, HeLa Cells, Molecular Chaperones
Abstract

R2TP is an HSP90 co-chaperone that assembles important macro-molecular machineries. It is composed of an RPAP3-PIH1D1 heterodimer, which binds the two essential AAA+ATPases RUVBL1/RUVBL2. Here, we resolve the structure of the conserved C-terminal domain of RPAP3, and we show that it directly binds RUVBL1/RUVBL2 hexamers. The human genome encodes two other proteins bearing RPAP3-C-terminal-like domains and three containing PIH-like domains. Systematic interaction analyses show that one RPAP3-like protein, SPAG1, binds PIH1D2 and RUVBL1/2 to form an R2TP-like complex termed R2SP. This co-chaperone is enriched in testis and among 68 of the potential clients identified, some are expressed in testis and others are ubiquitous. One substrate is liprin-α2, which organizes large signaling complexes. Remarkably, R2SP is required for liprin-α2 expression and for the assembly of liprin-α2 complexes, indicating that R2SP functions in quaternary protein folding. Effects are stronger at 32 °C, suggesting that R2SP could help compensating the lower temperate of testis., R2TP is an HSP90 co-chaperone composed of an RPAP3-PIH1D1 heterodimer, which binds two essential AAA+ ATPases RUVBL1/RUVBL2. Here authors use a structural approach to study RPAP3 and find an RPAP3-like protein (SPAG1) which also forms a co-chaperone complex with PIH1D2 and RUVBL1/2 enriched in testis.

Published
2018

16. The small nucleolar RNA U14 adopts a 'weak' K-turn conformation in solution

Author

Chagot, Marie-Eve, Quinternet, Marc, Rothé, Benjamin, Coutant, Jérôme, Charpentier, Bruno, Manival, Xavier, Isabelle, Lebars, Ingénierie Moléculaire et Physiopathologie Articulaire (IMoPA), Centre National de la Recherche Scientifique (CNRS)-Université de Lorraine (UL), Ingénierie, Biologie et Santé en Lorraine (IBSLor), Université de Lorraine (UL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Ecole Polytechnique Fédérale de Lausanne (EPFL), Bruker France, Institut de biologie moléculaire et cellulaire (IBMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS), and MANIVAL, Xavier

Subjects
[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, ComputingMilieux_MISCELLANEOUS
Abstract

International audience

Published
2018

17. Structure-function analysis of the box C/D snoRNP assembly factor Bcd1p

Author

Bragantini, Benoit, Paul, Arnaud, Charron, Christophe, Tiotiu, Decebal, Quinternet, Marc, Rothé, Benjamin, Chagot, Marie-Eve, Marty, Hélène, Massenet, Séverine, Saveanu, Cosmin, Labialle, Stéphane, Manival, Xavier, Charpentier, Bruno, and Charpentier, Bruno

Subjects
[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology
Abstract

In eukaryotes, numerous trans-acting factors orchestrate the various steps of ribosome biogenesis, among which hundreds of box C/D and box H/ACA small nucleolar ribonucleoproteins (snoRNPs) are catalysts of rRNA 2'-O-methylation and pseudouridylation, respectively. In addition, a subset of snoRNPs, e.g. U3, is required for endo-ribonucleolytic cleavages in pre-rRNAs. C/D snoRNPs each contain a set of four core proteins (Snu13, Nop58, Nop56, Nop1) and a specific non coding C/D snoRNA which contains guide sequences that base-pair with a target sequence to specify the site to modify. Assembly of C/D snoRNPs is a complex process involving the HSP90 co-chaperone R2TP which is composed of an RPAP3/Tah1p-PIH1D1/Pih1p heterodimer associated with the two essential AAA+ ATPases RUVBL1/Rvb1p/Pontin and RUVBL2/Rvb2p/Reptin (human/yeast). The platform protein NUFIP1/Rsa1p plays a central role in R2TP recruitment on pre-snoRNPs. Finally, protein ZNHIT6/Bcd1 appeared as a leading actor of the assembly process but its precise function is poorly characterized. By chromatin, RNA and protein immunoprecipitation assays we show that Bcd1p is recruited co-transcriptionally on pre-snoRNAs and directs the loading of the core protein Nop58 on RNA. We found unexpectedly that Bcd1p is able to interact with histone chaperone Rtt106p, which is involved in nucleosome formation associated with replication and in the control of heterochromatin formation. We report the structural characterization of the Bcd1p-Rtt106p interaction by X-ray crystallography and a combination of biophysical techniques. Moreover, we found Rtt106p associated with C/D snoRNAs in a Bcd1p- and Rsa1p-dependent manner. This interaction may highlight a functional relation between snoRNPs biogenesis and chromatin remodeling.

Published
2018

18. Box C/D snoRNPs: solid-state NMR fingerprint of an early-stage 50 kDa assembly inermediate

Author

Chagot, Marie-Eve, Quinternet, Marc, Manival, Xavier, Gardiennet, Carole, Gardiennet, Carole, Ingénierie Moléculaire et Physiopathologie Articulaire (IMoPA), Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS), Ingénierie, Biologie et Santé en Lorraine (IBSLor), Université de Lorraine (UL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Cristallographie, Résonance Magnétique et Modélisations (CRM2), and Centre National de la Recherche Scientifique (CNRS)-Université de Lorraine (UL)

Subjects
[SDV] Life Sciences [q-bio], [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], [SDV]Life Sciences [q-bio], [CHIM] Chemical Sciences, [CHIM]Chemical Sciences
Abstract

International audience; Many cellular functions rely on stable protein-only or protein-RNA complexes. Deciphering their assembly mechanism is a key question in cell biology.We here focus on box C/D snoRNPs (Ribo Nucleo Proteins), involved in ribosome biogenesis and present in archae, yeast and humans. Despite their relatively simple composition – one snoRNA and 4 core proteins – these particles don't self-assemble and their formation requires a large number of other proteins, called assembly factors.We use solid-state NMR to get atomic-scale information on the early part of Saccharomyces cerevisiae box C/D snoRNP assembly pathway, through the study of a protein complex composed of Snu13p core protein and Rsa1p:Hit1p assembly factors.Spectra of uniformly 13C,15N labeled Snu13p co-sedimented with unlabeled Rsa1p:Hit1p show good linewidths.We present first solid-state 13C and 15N assignments of the 126-residue protein Snu13p in the context of the 50 kDa complex. They are compared with solution chemical shifts of both the isolated protein and the protein in the presence of a 22-residue peptide from Rsa1p. As expected, the overall conformation of Snu13p is retained in the complex, but some residues experience significant chemical shift perturbations, an indicator of local conformational changes upon interaction with Rsa1p and Hit1p.Besides structural and conformational details, we could obtain the spectroscopic fingerprint of this complex, setting the basis for further studies, which will involve other partners of Snu13p:Rsa1p:Hit1p, as snoRNA.

Published
2018

22. Deep Structural Analysis of RPAP3 and PIH1D1, Two Components of the HSP90 Co-chaperone R2TP Complex

Author

Henri, Julien, primary, Chagot, Marie-Eve, additional, Bourguet, Maxime, additional, Abel, Yoann, additional, Terral, Guillaume, additional, Maurizy, Chloé, additional, Aigueperse, Christelle, additional, Georgescauld, Florian, additional, Vandermoere, Franck, additional, Saint-Fort, Rénette, additional, Behm-Ansmant, Isabelle, additional, Charpentier, Bruno, additional, Pradet-Balade, Bérengère, additional, Verheggen, Céline, additional, Bertrand, Edouard, additional, Meyer, Philippe, additional, Cianférani, Sarah, additional, Manival, Xavier, additional, and Quinternet, Marc, additional

Published
2018
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23. Structural dynamics and single-stranded DNA binding activity of the three N-Terminal domains of the large subunit of replication protein A from small angle X-ray scattering

Author

Pretto, Dalyir I., Tsutakawa, Susan, Brosey, Chris A., Castillo, Amalchi, Chagot, Marie-Eve, Smith, Jarrod A., Tainer, John A., and Chazin, Walter J.

Subjects
DNA-ligand interactions -- Analysis, Globular proteins -- Structure, Globular proteins -- Chemical properties, Biological sciences, Chemistry
Abstract

Small angle X-ray scattering (SAXS) experiments with two multidomain constructs from the N-terminus of the large subunit (RPA70) were used to examine the structural dynamics of these domains and their response to the binding of ssDNA. The obtained results supported a model in which RPA70N remains structurally independent of RPA70AB in the DNA-bound state and hence freely available to serve as a protein recruitment module.

Published
2010

24. NMR analysis of the architecture and functional remodeling of a modular multidomain protein, RPA

Author

Brosey, Chris A., Chagot, Marie-Eve, Ehrhardt, Mark, Pretto, Dalyir I., Weiner, Brian E., and Chazin, Walter J.

Subjects
DNA binding proteins -- Structure, DNA binding proteins -- Chemical properties, DNA replication -- Analysis, Nuclear magnetic resonance spectroscopy -- Usage, Phosphorylation -- Analysis, Chemistry
Abstract

The eukaryotic ssDNA binding protein, Replication Protein A (RPA) is used to demonstrate the application of NMR spectroscopy as a powerfull tool to characterize the remodeling of architecture in different functional states of modular proteins with multiple domains. The first direct evidence obtained for the remodeling of RPA upon binding ssDNA, including an alteration in the availability of the RPA32N domain might help explain damage-dependent phosphorylation in modular proteins.

Published
2009

26. Structure/Function Analysis of Protein–Protein Interactions Developed by the Yeast Pih1 Platform Protein and Its Partners in Box C/D snoRNP Assembly

Author

Quinternet, Marc, primary, Rothé, Benjamin, additional, Barbier, Muriel, additional, Bobo, Claude, additional, Saliou, Jean-Michel, additional, Jacquemin, Clémence, additional, Back, Régis, additional, Chagot, Marie-Eve, additional, Cianférani, Sarah, additional, Meyer, Philippe, additional, Branlant, Christiane, additional, Charpentier, Bruno, additional, and Manival, Xavier, additional

Published
2015
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27. Proteomic and 3D structure analyses highlight the C/D box snoRNP assembly mechanism and its control

Author

Bizarro, Jonathan, primary, Charron, Christophe, additional, Boulon, Séverine, additional, Westman, Belinda, additional, Pradet-Balade, Bérengère, additional, Vandermoere, Franck, additional, Chagot, Marie-Eve, additional, Hallais, Marie, additional, Ahmad, Yasmeen, additional, Leonhardt, Heinrich, additional, Lamond, Angus, additional, Manival, Xavier, additional, Branlant, Christiane, additional, Charpentier, Bruno, additional, Verheggen, Céline, additional, and Bertrand, Edouard, additional

Published
2014
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31. NOPCHAP1 is a PAQosome cofactor that helps loading NOP58 on RUVBL1/2 during box C/D snoRNP biogenesis.

Author

Abel Y, Paiva ACF, Bizarro J, Chagot ME, Santo PE, Robert MC, Quinternet M, Vandermoere F, Sousa PMF, Fort P, Charpentier B, Manival X, Bandeiras TM, Bertrand E, and Verheggen C

Subjects
Adenosine Triphosphate metabolism, Eye Proteins metabolism, Gene Knockout Techniques, Genes, Reporter, HSP90 Heat-Shock Proteins metabolism, HeLa Cells, Humans, Multiprotein Complexes, Protein Domains, Protein Interaction Mapping, Proteomics methods, Recombinant Fusion Proteins metabolism, Ribonucleoproteins, Small Nucleolar metabolism, Saccharomyces cerevisiae Proteins metabolism, ATPases Associated with Diverse Cellular Activities metabolism, Carrier Proteins metabolism, DNA Helicases metabolism, Molecular Chaperones metabolism, Nuclear Proteins metabolism, Ribonucleoproteins, Small Nucleolar biosynthesis
Abstract

The PAQosome is a large complex composed of the HSP90/R2TP chaperone and a prefoldin-like module. It promotes the biogenesis of cellular machineries but it is unclear how it discriminates closely related client proteins. Among the main PAQosome clients are C/D snoRNPs and in particular their core protein NOP58. Using NOP58 mutants and proteomic experiments, we identify different assembly intermediates and show that C12ORF45, which we rename NOPCHAP1, acts as a bridge between NOP58 and PAQosome. NOPCHAP1 makes direct physical interactions with the CC-NOP domain of NOP58 and domain II of RUVBL1/2 AAA+ ATPases. Interestingly, NOPCHAP1 interaction with RUVBL1/2 is disrupted upon ATP binding. Moreover, while it robustly binds both yeast and human NOP58, it makes little interactions with NOP56 and PRPF31, two other closely related CC-NOP proteins. Expression of NOP58, but not NOP56 or PRPF31, is decreased in NOPCHAP1 KO cells. We propose that NOPCHAP1 is a client-loading PAQosome cofactor that selects NOP58 to promote box C/D snoRNP assembly., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Published
2021
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32. Application of NMR Spectroscopy to Determine the 3D Structure of Small Non-Coding RNAs.

Author

Chagot ME, Quinternet M, Manival X, and Lebars I

Subjects
Nuclear Magnetic Resonance, Biomolecular, Nucleic Acid Conformation, RNA, Fungal chemistry, RNA, Fungal metabolism, RNA, Small Untranslated metabolism, RNA, Small Untranslated chemistry, Saccharomyces cerevisiae genetics
Abstract

Many RNA architectures were discovered to be involved in a wide range of essential biological processes in all organisms from carrying genetic information to gene expression regulation. The remarkable ability of RNAs to adopt various architectures depending on their environment enables the achievement of their myriads of biological functions. Nuclear Magnetic Resonance (NMR) is a powerful technique to investigate both their structure and dynamics. NMR is also a key tool for studying interactions between RNAs and their numerous partners such as small molecules, ions, proteins, or other nucleic acids.In this chapter, to illustrate the use of NMR for 3D structure determination of small noncoding RNA, we describe detailed methods that we used for the yeast C/D box small nucleolar RNA U14 from sample preparation to 3D structure calculation.

Published
2021
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33. The RPAP3-Cterminal domain identifies R2TP-like quaternary chaperones.

Author

Maurizy C, Quinternet M, Abel Y, Verheggen C, Santo PE, Bourguet M, C F Paiva A, Bragantini B, Chagot ME, Robert MC, Abeza C, Fabre P, Fort P, Vandermoere F, M F Sousa P, Rain JC, Charpentier B, Cianférani S, Bandeiras TM, Pradet-Balade B, Manival X, and Bertrand E

Subjects
Adaptor Proteins, Signal Transducing metabolism, Antigens, Surface metabolism, Apoptosis Regulatory Proteins genetics, Carrier Proteins genetics, Cell Line, GTP-Binding Proteins metabolism, HEK293 Cells, HeLa Cells, Humans, Male, Membrane Proteins metabolism, Protein Binding, Protein Folding, Protein Structure, Secondary, Signal Transduction, ATPases Associated with Diverse Cellular Activities metabolism, Apoptosis Regulatory Proteins metabolism, Carrier Proteins metabolism, DNA Helicases metabolism, HSP90 Heat-Shock Proteins metabolism, Molecular Chaperones metabolism, Testis metabolism
Abstract

R2TP is an HSP90 co-chaperone that assembles important macro-molecular machineries. It is composed of an RPAP3-PIH1D1 heterodimer, which binds the two essential AAA+ATPases RUVBL1/RUVBL2. Here, we resolve the structure of the conserved C-terminal domain of RPAP3, and we show that it directly binds RUVBL1/RUVBL2 hexamers. The human genome encodes two other proteins bearing RPAP3-C-terminal-like domains and three containing PIH-like domains. Systematic interaction analyses show that one RPAP3-like protein, SPAG1, binds PIH1D2 and RUVBL1/2 to form an R2TP-like complex termed R2SP. This co-chaperone is enriched in testis and among 68 of the potential clients identified, some are expressed in testis and others are ubiquitous. One substrate is liprin-α2, which organizes large signaling complexes. Remarkably, R2SP is required for liprin-α2 expression and for the assembly of liprin-α2 complexes, indicating that R2SP functions in quaternary protein folding. Effects are stronger at 32 °C, suggesting that R2SP could help compensating the lower temperate of testis.

Published
2018
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34. Preparation of the modular multi-domain protein RPA for study by NMR spectroscopy.

Author

Brosey CA, Chagot ME, and Chazin WJ

Subjects
Cell Culture Techniques methods, Cell Transformation, Viral, Chromatography, Gel, Culture Media chemistry, DNA, Single-Stranded metabolism, Isotope Labeling methods, Protein Structure, Tertiary, Replication Protein A metabolism, Nuclear Magnetic Resonance, Biomolecular methods, Protein Conformation, Replication Protein A biosynthesis, Replication Protein A chemistry, Replication Protein A isolation & purification
Abstract

The integrity and propagation of the genome depend upon the fidelity of DNA processing events, such as replication, damage recognition, and repair. Requisite to the numerous biochemical tasks required for DNA processing is the generation and manipulation of single-stranded DNA (ssDNA). As the primary eukaryotic ssDNA-binding protein, Replication Protein A (RPA) protects ssDNA templates from stray nuclease cleavage and untimely reannealment. More importantly, RPA also serves as a platform for organizing access to ssDNA for readout of the genetic code, recognition of aberrations in DNA, and processing by enzymes. We have proposed that RPA's ability to adapt to such a broad spectrum of multiprotein machinery arises in part from its modular organization and interdomain flexibility. While requisite for function, RPA's modular flexibility has presented many challenges to providing a detailed characterization of the dynamic architecture of the full-length protein. To enable the study of RPA's interdomain dynamics and responses to ssDNA binding by biophysical methods including NMR spectroscopy, we have successfully produced recombinant full-length RPA in milligram quantities at natural abundance and enriched with NMR-active isotopes.

Published
2012
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