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.
Characterization of the interaction between protein Snu13p/15.5K and the Rsa1p/NUFIP factor and demonstration of its functional importance for snoRNP assembly
Rothe, B.; Back, R.; Quinternet, M.; Bizarro, J.; Robert, M. C.; Blaud, M.; Romier, C.; Manival*, X.; Charpentier*, B.; Bertrand*, E.; Branlant, C.
Nucleic Acids Res
2014-02 / vol 42 / pages 2015-36
1362-4962 (Electronic) 0305-1048 (Linking)
Humans; Amino Acid Sequence; Hydrophobic and Hydrophilic Interactions; Models, Molecular; Molecular Chaperones/metabolism; Molecular Sequence Data; Nuclear Proteins/metabolism; Protein Binding; Protein Interaction Domains and Motifs; Protein Structure, Secondary; Ribonucleoproteins, Small Nuclear/*chemistry/metabolism; Ribonucleoproteins, Small Nucleolar/*metabolism; Ribosomal Proteins/*chemistry/metabolism; RNA Stability; Saccharomyces cerevisiae Proteins/*chemistry/metabolism; Saccharomyces cerevisiae/genetics/growth & development; Static Electricity