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.

[1]  Alexandre M J J Bonvin,et al.  HADDOCK versus HADDOCK: New features and performance of HADDOCK2.0 on the CAPRI targets , 2007, Proteins.

[2]  H. Urlaub,et al.  Hierarchical, clustered protein interactions with U4/U6 snRNA: a biochemical role for U4/U6 proteins , 2002, The EMBO journal.

[3]  Jinzhong Lin,et al.  Structural organization of box C/D RNA-guided RNA methyltransferase , 2009, Proceedings of the National Academy of Sciences.

[4]  P. Carbon,et al.  The SBP2 and 15.5 kD/Snu13p proteins share the same RNA binding domain: identification of SBP2 amino acids important to SECIS RNA binding. , 2002, RNA.

[5]  Hong Li,et al.  Structural comparison of yeast snoRNP and spliceosomal protein Snu13p with its homologs. , 2005, Biochemical and biophysical research communications.

[6]  C. Dominguez,et al.  HADDOCK: a protein-protein docking approach based on biochemical or biophysical information. , 2003, Journal of the American Chemical Society.

[7]  U. Fischer,et al.  Deciphering the assembly pathway of Sm‐class U snRNPs , 2008, FEBS letters.

[8]  Jinzhong Lin,et al.  Structural basis for site-specific ribose methylation by box C/D RNA protein complexes , 2011, Nature.

[9]  R. Lührmann,et al.  Conserved Stem II of the Box C/D Motif Is Essential for Nucleolar Localization and Is Required, Along with the 15.5K Protein, for the Hierarchical Assembly of the Box C/D snoRNP , 2002, Molecular and Cellular Biology.

[10]  W. Houry,et al.  Hsp90: a chaperone for protein folding and gene regulation. , 2005, Biochemistry and cell biology = Biochimie et biologie cellulaire.

[11]  Elizabeth J. Tran,et al.  Efficient RNA 2′‐O‐methylation requires juxtaposed and symmetrically assembled archaeal box C/D and C′/D′ RNPs , 2003, The EMBO journal.

[12]  A. Omer,et al.  In vitro reconstitution and activity of a C/D box methylation guide ribonucleoprotein complex , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[13]  N. Watkins,et al.  Evidence that the AAA+ Proteins TIP48 and TIP49 Bridge Interactions between 15.5K and the Related NOP56 and NOP58 Proteins during Box C/D snoRNP Biogenesis , 2009, Molecular and Cellular Biology.

[14]  F. Vignols,et al.  AtNUFIP, an essential protein for plant development, reveals the impact of snoRNA gene organisation on the assembly of snoRNPs and rRNA methylation in Arabidopsis thaliana. , 2011, The Plant journal : for cell and molecular biology.

[15]  G. Pruijn,et al.  The hU3-55K Protein Requires 15.5K Binding to the Box B/C Motif as Well as Flanking RNA Elements for Its Association with the U3 Small Nucleolar RNA in Vitro * , 2002, The Journal of Biological Chemistry.

[16]  B. Carlson,et al.  A novel RNA binding protein, SBP2, is required for the translation of mammalian selenoprotein mRNAs , 2000, The EMBO journal.

[17]  chen wang,et al.  Architecture and assembly of mammalian H/ACA small nucleolar and telomerase ribonucleoproteins , 2004, The EMBO journal.

[18]  W. Delano.  The PyMOL Molecular Graphics System , 2002 .

[19]  R. Terns,et al.  Non-coding RNAs: lessons from the small nuclear and small nucleolar RNAs , 2007, Nature Reviews Molecular Cell Biology.

[20]  U. Fischer,et al.  The role of RNP biogenesis in spinal muscular atrophy. , 2009, Current opinion in cell biology.

[21]  Alexander D. MacKerell,et al.  Development and current status of the CHARMM force field for nucleic acids , 2000, Biopolymers.

[22]  K. Hartmuth,et al.  RNA Structural Requirements for the Association of the Spliceosomal hPrp31 Protein with the U4 and U4atac Small Nuclear Ribonucleoproteins* , 2006, Journal of Biological Chemistry.

[23]  M. Caizergues-Ferrer,et al.  Nhp2p and Nop10p are essential for the function of H/ACA snoRNPs , 1998, The EMBO journal.

[24]  S. Moréra,et al.  The Pih1-Tah1 Cochaperone Complex Inhibits Hsp90 Molecular Chaperone ATPase Activity* , 2010, The Journal of Biological Chemistry.

[25]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[26]  G. Dreyfuss,et al.  The SMN Complex Is Associated with snRNPs throughout Their Cytoplasmic Assembly Pathway , 2002, Molecular and Cellular Biology.

[27]  Tamás Kiss,et al.  Modification of Sm small nuclear RNAs occurs in the nucleoplasmic Cajal body following import from the cytoplasm , 2003, The EMBO journal.

[28]  A. Fatica,et al.  The Cotranscriptional Assembly of snoRNPs Controls the Biosynthesis of H/ACA snoRNAs in Saccharomyces cerevisiae , 2005, Molecular and Cellular Biology.

[29]  A. Cléry,et al.  A structural, phylogenetic, and functional study of 15.5-kD/Snu13 protein binding on U3 small nucleolar RNA. , 2003, RNA.

[30]  Tamás Kiss,et al.  Cajal body‐specific small nuclear RNAs: a novel class of 2′‐O‐methylation and pseudouridylation guide RNAs , 2002, The EMBO journal.

[31]  K. Hartmuth,et al.  Crystal structure of the spliceosomal 15.5kD protein bound to a U4 snRNA fragment. , 2000, Molecular cell.

[32]  G. Bitter,et al.  Expression of heterologous genes in Saccharomyces cerevisiae from vectors utilizing the glyceraldehyde-3-phosphate dehydrogenase gene promoter. , 1984, Gene.

[33]  Christiane Branlant,et al.  A Common Core RNP Structure Shared between the Small Nucleoar Box C/D RNPs and the Spliceosomal U4 snRNP , 2000, Cell.

[34]  C. Branlant,et al.  Analysis of Sequence and Structural Features That Identify the B/C Motif of U3 Small Nucleolar RNA as the Recognition Site for the Snu13p-Rrp9p Protein Pair , 2006, Molecular and Cellular Biology.

[35]  X. Darzacq,et al.  Stepwise RNP assembly at the site of H/ACA RNA transcription in human cells , 2006, The Journal of cell biology.

[36]  D. Kressler,et al.  Synthetic Lethality with Conditional dbp6 Alleles Identifies Rsa1p, a Nucleoplasmic Protein Involved in the Assembly of 60S Ribosomal Subunits , 1999, Molecular and Cellular Biology.

[37]  B. Bardoni,et al.  The Hsp90 chaperone controls the biogenesis of L7Ae RNPs through conserved machinery , 2008, The Journal of cell biology.

[38]  C. Will,et al.  The Spliceosome: Design Principles of a Dynamic RNP Machine , 2009, Cell.

[39]  A. Lamond,et al.  HSP90 and its R2TP/Prefoldin-like cochaperone are involved in the cytoplasmic assembly of RNA polymerase II. , 2010, Molecular cell.

[40]  D. Moras,et al.  Dissecting the interaction network of multiprotein complexes by pairwise coexpression of subunits in E. coli. , 2001, Journal of molecular biology.

[41]  J. Yong,et al.  Essential Role for the SMN Complex in the Specificity of snRNP Assembly , 2002, Science.

[42]  T. Steitz,et al.  The complete atomic structure of the large ribosomal subunit at 2.4 A resolution. , 2000, Science.

[43]  T. Kiss.  Small Nucleolar RNAs An Abundant Group of Noncoding RNAs with Diverse Cellular Functions , 2002, Cell.

[44]  R. Terns,et al.  Structural basis for substrate placement by an archaeal box C/D ribonucleoprotein particle. , 2011, Molecular cell.

[45]  Laxmikant V. Kalé,et al.  Scalable molecular dynamics with NAMD , 2005, J. Comput. Chem..

[46]  E. Bertrand,et al.  A Dynamic Scaffold of Pre-snoRNP Factors Facilitates Human Box C/D snoRNP Assembly , 2007, Molecular and Cellular Biology.

[47]  P. Güntert.  Automated NMR structure calculation with CYANA. , 2004, Methods in molecular biology.

[48]  Shuang Li,et al.  Structure of the Shq1–Cbf5–Nop10–Gar1 complex and implications for H/ACA RNP biogenesis and dyskeratosis congenita , 2011, The EMBO journal.

[49]  M. Fournier,et al.  Depletion of U14 small nuclear RNA (snR128) disrupts production of 18S rRNA in Saccharomyces cerevisiae. , 1990, Molecular and cellular biology.

[50]  David Tollervey,et al.  Making ribosomes. , 2002, Current opinion in cell biology.

[51]  H. Urlaub,et al.  Functional interaction of a novel 15.5kD[U4/U6·U5] tri‐snRNP protein with the5′ stem–loop of U4 snRNA , 1999, The EMBO journal.

[52]  C. Branlant,et al.  Reconstitution of archaeal H/ACA small ribonucleoprotein complexes active in pseudouridylation , 2005, Nucleic acids research.

[53]  F. Allain,et al.  High-resolution structural analysis shows how Tah1 tethers Hsp90 to the R2TP complex. , 2013, Structure.

[54]  Wayne A. Decatur,et al.  New bioinformatic tools for analysis of nucleotide modifications in eukaryotic rRNA. , 2007, RNA.

[55]  A. Bonvin,et al.  WHISCY: What information does surface conservation yield? Application to data‐driven docking , 2006, Proteins.

[56]  C. Sander,et al.  A novel RNA-binding motif in omnipotent suppressors of translation termination, ribosomal proteins and a ribosome modification enzyme? , 1994, Nucleic acids research.

[57]  T. Kiss,et al.  Box H/ACA small ribonucleoproteins. , 2010, Molecular cell.

[58]  R. Raz,et al.  ProMate: a structure based prediction program to identify the location of protein-protein binding sites. , 2004, Journal of molecular biology.

[59]  Christophe Romier,et al.  Co-expression of protein complexes in prokaryotic and eukaryotic hosts: experimental procedures, database tracking and case studies. , 2006, Acta crystallographica. Section D, Biological crystallography.

[60]  Daniel L Baker,et al.  RNA-guided RNA modification: functional organization of the archaeal H/ACA RNP. , 2005, Genes & development.

[61]  Thomas Ried,et al.  From Silencing to Gene Expression Real-Time Analysis in Single Cells , 2004, Cell.

[62]  A. Brunger.  Version 1.2 of the Crystallography and NMR system , 2007, Nature Protocols.

[63]  E. Blackburn,et al.  Telomerase: an RNP enzyme synthesizes DNA. , 2011, Cold Spring Harbor perspectives in biology.

[64]  T. Hughes,et al.  Molecular chaperone Hsp90 stabilizes Pih1/Nop17 to maintain R2TP complex activity that regulates snoRNA accumulation , 2008, The Journal of cell biology.

[65]  C. Romier,et al.  Deciphering correct strategies for multiprotein complex assembly by co-expression: application to complexes as large as the histone octamer. , 2011, Journal of structural biology.

[66]  R. Lührmann,et al.  Protein-Protein and Protein-RNA Contacts both Contribute to the 15.5K-Mediated Assembly of the U4/U6 snRNP and the Box C/D snoRNPs , 2006, Molecular and Cellular Biology.

[67]  G. Varani,et al.  The H/ACA RNP assembly factor SHQ1 functions as an RNA mimic. , 2011, Genes & development.

[68]  Ping Li,et al.  Binding of the Human Prp31 Nop Domain to a Composite RNA-Protein Platform in U4 snRNP , 2007, Science.

[69]  David S. Wishart,et al.  PREDITOR: a web server for predicting protein torsion angle restraints , 2006, Nucleic Acids Res..

[70]  G. Varani,et al.  The box H/ACA RNP assembly factor Naf1p contains a domain homologous to Gar1p mediating its interaction with Cbf5p. , 2007, Journal of molecular biology.