Advertisement

Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

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

Abstract

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 small nucleolar ribonucleoproteins involved in ribosome biogenesis. The mature particles contain four core proteins and a guide RNA. Despite their relatively simple composition, these particles don't self-assemble in eukaryote and the production of a native and functional particle requires a large number of transient other proteins, called assembly factors. We present here 13C and 15N solid-state NMR assignment of yeast 126-residue core protein Snu13 in the context of its 50 kDa pre-complex with assembly factors Rsa1p:Hit1p. In this sample, only one third of the protein is labelled, leading to a low sensitivity. We could nevertheless obtain assignment data for 91% of the residues. Secondary structure derived from our assignments shows that Snu13p overall structure is maintained in the context of the complex. Chemical shift perturbations are analysed to evaluate Snu13p conformational changes and interaction interface upon binding to its partner proteins. While indirect perturbations are observed in the hydrophobic core, we find other good candidate residues belonging to the interaction interface. We describe the role of some Snu13p N-terminal and C-terminal residues, not identified in previous structural studies. These preliminary results will serve as a basis for future interaction studies, especially by adding RNA, to decipher box C/D snoRNP particles assembly pathway.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  1. Bachellerie J-P, Cavaillé J, Hüttenhofer A (2002) The expanding snoRNA world. Biochimie 84:775–790. https://doi.org/10.1016/S0300-9084(02)01402-5

  2. Bizarro J, Charron C, Boulon S, Westman B, Pradet-Balade B, Vandermoere F, Chagot M-E, Hallais M, Ahmad Y, Leonhardt H, Lamond A, Manival X, Branlant C, Charpentier B, Verheggen C, Bertrand E (2014) Proteomic and 3D structure analyses highlight the C/D box snoRNP assembly mechanism and its control. J Cell Biol 207:463–480. https://doi.org/10.1083/jcb.201404160

  3. Böckmann A, Gardiennet C, Verel R, Hunkeler A, Loquet A, Pintacuda G, Emsley L, Meier BH, Lesage A (2009) Characterization of different water pools in solid-state NMR protein samples. J Biomol NMR 45:319–327. https://doi.org/10.1007/s10858-009-9374-3

  4. Boulon S, Marmier-Gourrier N, Pradet-Balade B, Wurth L, Verheggen C, Jády BE, Rothé B, Pescia C, Robert M-C, Kiss T, Bardoni B, Krol A, Branlant C, Allmang C, Bertrand E, Charpentier B (2008) The Hsp90 chaperone controls the biogenesis of L7Ae RNPs through conserved machinery. J Cell Biol 180:579–595. https://doi.org/10.1083/jcb.200708110

  5. Chagot M-E, Quinternet M, Rothé B, Charpentier B, Coutant J, Manival X, Lebars I (2019) The yeast C/D box snoRNA U14 adopts a “weak” K-turn like conformation recognized by the Snu13 core protein in solution. Biochimie 164:70–82. https://doi.org/10.1016/j.biochi.2019.03.014

  6. Dobbyn HC, McEwan PA, Krause A, Novak-Frazer L, Bella J, O’Keefe RT (2007) Analysis of pre-mRNA and pre-rRNA processing factor Snu13p structure and mutants. Biochem Biophys Res Commun 360:857–862. https://doi.org/10.1016/j.bbrc.2007.06.163

  7. Habenstein B, Wasmer C, Bousset L, Sourigues Y, Schütz A, Loquet A, Meier BH, Melki R, Böckmann A (2011) Extensive de novo solid-state NMR assignments of the 33 kDa C-terminal domain of the Ure2 prion. J Biomol NMR 51:235–243. https://doi.org/10.1007/s10858-011-9530-4

  8. Kabsch W, Sander C (1983) Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers 22:2577–2637. https://doi.org/10.1002/bip.360221211

  9. Keller R (2004) The computer aided resonance assignment tutorial. Cantina Verl., Goldau

  10. Lafontaine DLJ (2015) Noncoding RNAs in eukaryotic ribosome biogenesis and function. Nat Struct Mol Biol 22:11–19. https://doi.org/10.1038/nsmb.2939

  11. Maiti R, Van Domselaar GH, Zhang H, Wishart DS (2004) SuperPose: a simple server for sophisticated structural superposition. Nucleic Acids Res 32:W590–W594. https://doi.org/10.1093/nar/gkh477

  12. Paul A, Tiotiu D, Bragantini B, Marty H, Charpentier B, Massenet S, Labialle S (2019) Bcd1p controls RNA loading of the core protein Nop58 during C/D box snoRNP biogenesis. RNA 25:496–506. https://doi.org/10.1261/rna.067967.118

  13. Peng Y, Yu G, Tian S, Li H (2014) Co-expression and Co-purification of archaeal and eukaryal box C/D RNPs. PLoS ONE 9:e103096. https://doi.org/10.1371/journal.pone.0103096

  14. Quinternet M, Rothé B, Barbier M, Bobo C, Saliou J-M, Jacquemin C, Back R, Chagot M-E, Cianférani S, Meyer P, Branlant C, Charpentier B, Manival X (2015) Structure/function analysis of protein-protein interactions developed by the yeast Pih1 platform protein and its partners in box C/D snoRNP assembly. J Mol Biol 427:2816–2839. https://doi.org/10.1016/j.jmb.2015.07.012

  15. Quinternet M, Chagot M-E, Rothé B, Tiotiu D, Charpentier B, Manival X (2016) Structural features of the box C/D snoRNPpre-assembly process are conserved through species. Structure 24:1693–1706. https://doi.org/10.1016/j.str.2016.07.016

  16. Rothé B, Back R, Quinternet M, Bizarro J, Robert M-C, Blaud M, Romier C, Manival X, Charpentier B, Bertrand E, Branlant C (2014a) Characterization of the interaction between protein Snu13p/15.5K and the Rsa1p/NUFIP factor and demonstration of its functional importance for snoRNP assembly. Nucleic Acids Res 42:2015–2036. https://doi.org/10.1093/nar/gkt1091

  17. Rothé B, Manival X, Rolland N, Charron C, Senty-Ségault V, Branlant C, Charpentier B (2017) Implication of the box C/D snoRNP assembly factor Rsa1p in U3 snoRNP assembly. Nucleic Acids Res 45:7455–7473. https://doi.org/10.1093/nar/gkx424

  18. Rothé B, Saliou J-M, Quinternet M, Back R, Tiotiu D, Jacquemin C, Loegler C, Schlotter F, Peña V, Eckert K, Morera S, Dorsselaer AV, Branlant C, Massenet S, Sanglier-Cianférani S, Manival X, Charpentier B (2014b) Protein Hit1, a novel box C/D snoRNP assembly factor, controls cellular concentration of the scaffolding protein Rsa1 by direct interaction. Nucleic Acids Res 42:10731–10747. https://doi.org/10.1093/nar/gku612

  19. Schuetz A, Wasmer C, Habenstein B, Verel R, Greenwald J, Riek R, Böckmann A, Meier BH (2010) Protocols for the sequential solid-state NMR spectroscopic assignment of a uniformly labeled 25 kDa protein: HET-s(1-227). Chem Bio Chem 11:1543–1551. https://doi.org/10.1002/cbic.201000124

  20. Schultz A, Nottrott S, Watkins NJ, Luhrmann R (2006) 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. Mol Cell Biol 26:5146–5154. https://doi.org/10.1128/MCB.02374-05

  21. Shen Y, Bax A (2013) Protein backbone and sidechain torsion angles predicted from NMR chemical shifts using artificial neural networks. J Biomol NMR 56:227–241. https://doi.org/10.1007/s10858-013-9741-y

  22. Stevens TJ, Fogh RH, Boucher W, Higman VA, Eisenmenger F, Bardiaux B, van Rossum B-J, Oschkinat H, Laue ED (2011) A software framework for analysing solid-state MAS NMR data. J Biomol NMR 51:437–447. https://doi.org/10.1007/s10858-011-9569-2

  23. Vranken WF, Boucher W, Stevens TJ, Fogh RH, Pajon A, Llinas M, Ulrich EL, Markley JL, Ionides J, Laue ED (2005) The CCPN data model for NMR spectroscopy: Development of a software pipeline. Proteins: Struct Funct Bioinf 59:687–696. https://doi.org/10.1002/prot.20449

  24. Wang YJ, Jardetzky O (2002) Probability-based protein secondary structure identification using combined NMR chemical-shift data. Protein Sci 11:852–861

  25. Watkins NJ, Dickmanns A, Luhrmann R (2002) 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. Mol Cell Biol 22:8342–8352. https://doi.org/10.1128/MCB.22.23.8342-8352.2002

  26. Williamson MP (2013) Using chemical shift perturbation to characterise ligand binding. Prog Nucl Magn Reson Spectrosc 73:1–16. https://doi.org/10.1016/j.pnmrs.2013.02.001

  27. Workman H, Skalicky JJ, Flynn PF (2008) Assignment of 1H, 13C, and 15N resonances of the RNA binding protein Snu13p from Saccharomyces cerevisiae. Biomolecular NMR Assignments 2:1–3. https://doi.org/10.1007/s12104-007-9069-1

Download references

Acknowledgements

Financial support from PEPS Mirabelle 2016 “ssNMRsnoRNP” and from the IR-RMN-THC Fr3050 CNRS for conducting the research is gratefully acknowledged.

Author information

Correspondence to Xavier Manival or Carole Gardiennet.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Electronic supplementary material 1 (DOCX 976 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Chagot, M., Quinternet, M., Jacquemin, C. et al. Box C/D snoRNPs: solid-state NMR fingerprint of an early-stage 50 kDa assembly intermediate. Biomol NMR Assign (2020). https://doi.org/10.1007/s12104-020-09933-y

Download citation

Keywords

  • Solid-state NMR
  • Saccharomyces cerevisiae
  • Box C/D snoRNP
  • Snu13p
  • Protein-protein interaction