Characterization of the interaction between protein Snu13p/15.5K and the Rsa1p/NUFIP factor and demonstration of its functional importance for snoRNP assembly.
The yeast Snu13p protein and its 15.5K human homolog both bind U4 snRNA and box C/D snoRNAs. They also bind the Rsa1p/NUFIP assembly factor, proposed to scaffold immature snoRNPs and to recruit the Hsp90-R2TP chaperone complex. However, the nature of the Snu13p/15.5K-Rsa1p/NUFIP interaction and its exact role in snoRNP assembly remained to be elucidated. By using biophysical, molecular and imaging approaches, here, we identify residues needed for Snu13p/15.5K-Rsa1p/NUFIP interaction. By NMR structure determination and docking approaches, we built a 3D model of the Snup13p-Rsa1p interface, suggesting that residues R249, R246 and K250 in Rsa1p and E72 and D73 in Snu13p form a network of electrostatic interactions shielded from the solvent by hydrophobic residues from both proteins and that residue W253 of Rsa1p is inserted in a hydrophobic cavity of Snu13p. Individual mutations of residues in yeast demonstrate the functional importance of the predicted interactions for both cell growth and snoRNP formation. Using archaeal box C/D sRNP 3D structures as templates, the association of Snu13p with Rsa1p is predicted to be exclusive of interactions in active snoRNPs. Rsa1p and NUFIP may thus prevent premature activity of pre-snoRNPs, and their removal may be a key step for active snoRNP production.
Ingénierie Moléculaire et Physiopathologie Articulaire (IMoPA), UMR 7365 CNRS Université de Lorraine, Biopôle de l'Université de Lorraine, Campus Biologie Santé, 9 avenue de la forêt de Haye, BP 184, 54505 Vandœuvre-lès-Nancy, France, FR CNRS-3209 (Ingénierie Moléculaire et Thérapeutique), CNRS, Université de Lorraine, Faculté de Médecine, Bâtiment Biopôle, BP 184, 54505 Vandœuvre-lès-Nancy Cedex, France, Equipe labellisée Ligue contre le Cancer, IGMM (Institut de Génétique Moléculaire de Montpellier), Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5535, Montpellier Cedex 5, France and IGBMC (Institut de Génétique et Biologie Moléculaire et Cellulaire), Département de Biologie et Génomique Structurales, Université de Strasbourg, CNRS, INSERM, 1 Rue Laurent Fries, BP 10142, 67404 Illkirch Cedex, France.
Benjamin
B
Rothé
Régis
R
Back
Marc
M
Quinternet
Jonathan
J
Bizarro
Marie-Cécile
MC
Robert
Magali
M
Blaud
Christophe
C
Romier
Xavier
X
Manival
Bruno
B
Charpentier
Edouard
E
Bertrand
Christiane
C
Branlant
D000595:Amino Acid Sequence; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000378:metabolism; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D020871:RNA Stability; D017411:Ribonucleoproteins, Small Nuclear / Q000737:chemistry* / Q000378:metabolism; D020718:Ribonucleoproteins, Small Nucleolar / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism; D055672:Static Electricity
D018832:Molecular Chaperones; D009687:Nuclear Proteins; C497702:PIH1 protein, S cerevisiae; C402485:RSA1 protein, S cerevisiae; D017411:Ribonucleoproteins, Small Nuclear; D020718:Ribonucleoproteins, Small Nucleolar; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins; C440569:Snu13 protein, S cerevisiae; C402291:Snu13 protein, human; C571197:Tah1 protein, S cerevisiae