Cryo-EM structure of an early precursor of large ribosomal subunit reveals a half-assembled intermediate

Abstract

Assembly of eukaryotic ribosome is a complicated and dynamic process that involves a series of intermediates. It is unknown how the highly intertwined structure of 60S large ribosomal subunits is established. Here, we report the structure of an early nucleolar pre-60S ribosome determined by cryo-electron microscopy at 3.7 Å resolution, revealing a half-assembled subunit. Domains I, II and VI of 25S/5.8S rRNA pack tightly into a native-like substructure, but domains III, IV and V are not assembled. The structure contains 12 assembly factors and 19 ribosomal proteins, many of which are required for early processing of large subunit rRNA. The Brx1-Ebp2 complex would interfere with the assembly of domains IV and V. Rpf1, Mak16, Nsa1 and Rrp1 form a cluster that consolidates the joining of domains I and II. Our structure reveals a key intermediate on the path to establishing the global architecture of 60S subunits.

Keywords

ribosome assembly / cryo-EM / pre-60S ribosome / nucleolar

References

[1] Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung LW, Kapral GJ, Grosse-Kunstleve RW (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66:213–221

[2] Amunts A, Brown A, Bai XC, Llacer JL, Hussain T, Emsley P, Long F, Murshudov G, Scheres SHW, Ramakrishnan V (2014) Structure of the yeast mitochondrial large ribosomal subunit. Science 343:1485–1489

[3] Asano N, Kato K, Nakamura A, Komoda K, Tanaka I, Yao M (2015) Structural and functional analysis of the Rpf2-Rrs1 complex in ribosome biogenesis. Nucleic Acids Res 43:4746–4757

[4] Barandun J, Chaker-Margot M, Hunziker M, Molloy KR, Chait BT, Klinge S (2017) The complete structure of the small-subunit processome. Nat Struct Mol Biol 24:944–953

[5] Barrio-Garcia C, Thoms M, Flemming D, Kater L, Berninghausen O, Bassler J, Beckmann R, Hurt E (2016) Architecture of the Rix1-Rea1 checkpoint machinery during pre-60S-ribosome remodeling. Nat Struct Mol Biol 23:37–44

[6] Bassler J, Kallas M, Pertschy B, Ulbrich C, Thoms M, Hurt E (2010) The AAA-ATPase Rea1 drives removal of biogenesis factors during multiple stages of 60S ribosome assembly. Mol Cell 38:712–721

[7] Bassler J, Ahmed YL, Kallas M, Kornprobst M, Calvino FR, Gnadig M, Thoms M, Stier G, Ismail S, Kharde S (2017) Interaction network of the ribosome assembly machinery from a eukaryotic thermophile. Protein Sci 26:327–342

[8] Ben-Shem A, Garreau de Loubresse N, Melnikov S, Jenner L, Yusupova G, Yusupov M (2011) The structure of the eukaryotic ribosome at 3.0 A resolution. Science 334:1524–1529

[9] Biasini M, Bienert S, Waterhouse A, Arnold K, Studer G, Schmidt T, Kiefer F, Gallo TCassarino M, Bertoni L, Bordoli (2014) SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res 42:W252–258

[10] Bradatsch B, Leidig C, Granneman S, Gnadig M, Tollervey D, Bottcher B, Beckmann R, Hurt E (2012) Structure of the pre-60S ribosomal subunit with nuclear export factor Arx1 bound at the exit tunnel. Nat Struct Mol Biol 19:1234–1241

[11] Brown A, Long F, Nicholls RA, Toots J, Emsley P, Murshudov G (2015) Tools for macromolecular model building and refinement into electron cryo-microscopy reconstructions. Acta Crystallogr D Biol Crystallogr 71:136–153

[12] Chaker-Margot M, Hunziker M, Barandun J, Dill BD, Klinge S (2015) Stage-specific assembly events of the 6-MDa small-subunit processome initiate eukaryotic ribosome biogenesis. Nat Struct Mol Biol 22:920–923

[13] Chaker-Margot, M., Barandun, J., Hunziker, M., and Klinge, S. (2017). Architecture of the yeast small subunit processome. Science 355, eaal1880.

[14] Chen W, Xie Z, Yang F, Ye K (2017) Stepwise assembly of the earliest precursors of large ribosomal subunits in yeast. Nucleic Acids Res 45:6837–6847

[15] Cheng J, Kellner N, Berninghausen O, Hurt E, Beckmann R (2017) 3.2-A-resolution structure of the 90S preribosome before A1 prerRNA cleavage. Nat Struct Mol Biol 24:954–964

[16] de la Cruz J, Karbstein K, Woolford JL Jr (2015) Functions of ribosomal proteins in assembly of eukaryotic ribosomes in vivo. Annu Rev Biochem 84:93–129

[17] Del Campo M, Lambowitz AM (2009) Structure of the Yeast DEAD box protein Mss116p reveals two wedges that crimp RNA. Mol Cell 35:598–609

[18] Dembowski JA, Kuo B, Woolford JL Jr (2013) Has1 regulates consecutive maturation and processing steps for assembly of 60S ribosomal subunits. Nucleic Acids Res 41:7889–7904

[19] Dragon F, Gallagher JE, Compagnone-Post PA, Mitchell BM, Porwancher KA, Wehner KA, Wormsley S, Settlage RE, Shabanowitz J, Osheim Y (2002) A large nucleolar U3 ribonucleoprotein required for 18S ribosomal RNA biogenesis. Nature 417:967–970

[20] Emery B, de la Cruz J, Rocak S, Deloche O, Linder P (2004) Has1p, a member of the DEAD-box family, is required for 40S ribosomal subunit biogenesis in Saccharomyces cerevisiae. Mol Microbiol 52:141–158

[21] Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60:2126–2132

[22] Erijman A, Dantes A, Bernheim R, Shifman JM, Peleg Y (2011) Transfer-PCR (TPCR): a highway for DNA cloning and protein engineering. J Struct Biol 175(2):171–177

[23] Falk S, Tants JN, Basquin J, Thoms M, Hurt E, Sattler M, Conti E (2017) Structural insights into the interaction of the nuclear exosome helicase Mtr4 with the preribosomal protein Nop53. RNA 23:1780–1787

[24] Fatica A, Cronshaw AD, Dlakic M, Tollervey D (2002) Ssf1p prevents premature processing of an early pre-60S ribosomal particle. Mol Cell 9:341–351

[25] Gamalinda M, Ohmayer U, Jakovljevic J, Kumcuoglu B, Woolford J, Mbom B, Lin L, Woolford JL Jr (2014) A hierarchical model for assembly of eukaryotic 60S ribosomal subunit domains. Genes Dev 28:198–210

[26] Grandi P, Rybin V, Bassler J, Petfalski E, Strauss D, Marzioch M, Schafer T, Kuster B, Tschochner H, Tollervey D (2002) 90S pre-ribosomes include the 35S pre-rRNA, the U3 snoRNP, and 40S subunit processing factors but predominantly lack 60S synthesis factors. Mol Cell 10:105–115

[27] Greber BJ, Gerhardy S, Leitner A, Leibundgut M, Salem M, Boehringer D, Leulliot N, Aebersold R, Panse VG, Ban N (2015) Insertion of the biogenesis factor Rei1 probes the ribosomal tunnel during 60S maturation. Cell 164:91–102

[28] Kater L, Thoms M, Barrio-Garcia C, Cheng J, Ismail S, Ahmed YL, Bange G, Kressler D, Berninghausen O, Sinning I (2017) Visualizing the assembly pathway of nucleolar Pre-60S ribosomes. Cell 171(1599–1610):e1514

[29] Kharde S, Calvino FR, Gumiero A, Wild K, Sinning I (2015) The structure of Rpf2-Rrs1 explains its role in ribosome biogenesis. Nucleic Acids Res 43:7083–7095

[30] Klinge S, Voigts-Hoffmann F, Leibundgut M, Arpagaus S, Ban N (2011) Crystal structure of the eukaryotic 60S ribosomal subunit in complex with initiation factor 6. Science 334:941–948

[31] Kornprobst M, Turk M, Kellner N, Cheng J, Flemming D, Kos-Braun I, Kos M, Thoms M, Berninghausen O, Beckmann R (2016) Architecture of the 90S pre-ribosome: a structural view on the birth of the eukaryotic ribosome. Cell 166:380–393

[32] Kressler D, Roser D, Pertschy B, Hurt E (2008) The AAA ATPase Rix7 powers progression of ribosome biogenesis by stripping Nsa1 from pre-60S particles. J Cell Biol 181:935–944

[33] Kucukelbir A, Sigworth FJ, Tagare HD (2014) Quantifying the local resolution of cryo-EM density maps. Nat Methods 11:63–65

[34] Leidig C, Thoms M, Holdermann I, Bradatsch B, Berninghausen O, Bange G, Sinning I, Hurt E, Beckmann R (2014) 60S ribosome biogenesis requires rotation of the 5S ribonucleoprotein particle. Nat Commun 5:3491

[35] Li X, Mooney P, Zheng S, Booth CR, Braunfeld MB, Gubbens S, Agard DA, Cheng Y (2013) Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat Methods 10:584–590

[36] Lo YH, Romes EM, Pillon MC, Sobhany M, Stanley RE (2017) Structural analysis reveals features of ribosome assembly factor Nsa1/WDR74 important for localization and interaction with Rix7/NVL2. Structure 25:762–772

[37] Ma C, Wu S, Li N, Chen Y, Yan K, Li Z, Zheng L, Lei J, Woolford JL Jr, Gao N (2017) Structural snapshot of cytoplasmic pre-60Sribosomal particles bound by Nmd3, Lsg1, Tif6 and Reh1. Nat Struct Mol Biol 24:214–220

[38] Madru C, Lebaron S, Blaud M, Delbos L, Pipoli J, Pasmant E, Rety S, Leulliot N (2015) Chaperoning 5S RNA assembly. Genes Dev 29:1432–1446

[39] Mallam AL, Del Campo M, Gilman B, Sidote DJ, Lambowitz AM (2012) Structural basis for RNA-duplex recognition and unwinding by the DEAD-box helicase Mss116p. Nature 490:121–125

[40] Malyutin AG, Musalgaonkar S, Patchett S, Frank J, Johnson AW (2017) Nmd3 is a structural mimic of eIF5A, and activates the cpGTPase Lsg1 during 60S ribosome biogenesis. EMBO J 36:854–868

[41] Mastronarde DN (2005) Automated electron microscope tomography using robust prediction of specimen movements. J Struct Biol 152:36–51

[42] Matsuo Y, Granneman S, Thoms M, Manikas RG, Tollervey D, Hurt E (2014) Coupled GTPase and remodelling ATPase activities form a checkpoint for ribosome export. Nature 505:112–116

[43] McCann KL, Charette JM, Vincent NG, Baserga SJ (2015) A protein interaction map of the LSU processome. Genes Dev 29:862–875

[44] Ng CL, Waterman D, Koonin EV, Antson AA, Ortiz-Lombardia M (2005) Crystal structure of Mil (Mth680): internal duplication and similarity between the Imp4/Brix domain and the anticodonbinding domain of class IIa aminoacyl-tRNA synthetases. EMBO Rep 6:140–146

[45] Osheim YN, French SL, Keck KM, Champion EA, Spasov K, Dragon F, Baserga SJ, Beyer AL (2004) Pre-18S ribosomal RNA is structurally compacted into the SSU processome prior to being cleaved from nascent transcripts in Saccharomyces cerevisiae. Mol Cell 16:943–954

[46] Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol 276:307–326

[47] Pena C, Hurt E, Panse VG (2017) Eukaryotic ribosome assembly, transport and quality control. Nat Struct Mol Biol 24:689–699

[48] Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612

[49] Popenda M, Szachniuk M, Antczak M, Purzycka KJ, Lukasiak P, Bartol N, Blazewicz J, Adamiak RW (2012) Automated 3D structure composition for large RNAs. Nucleic Acids Res 40:e112

[50] Rohou A, Grigorieff N (2015) CTFFIND4: fast and accurate defocus estimation from electron micrographs. J Struct Biol 192:216–221

[51] Sahasranaman A, Dembowski J, Strahler J, Andrews P, Maddock J, Woolford JL Jr (2011) Assembly of Saccharomyces cerevisiae 60S ribosomal subunits: role of factors required for 27S pre-rRNA processing. EMBO J 30:4020–4032

[52] Sa-Moura B, Kornprobst M, Kharde S, Ahmed YL, Stier G, Kunze R, Sinning I, Hurt E (2017) Mpp10 represents a platform for the interaction of multiple factors within the 90S pre-ribosome. PLoS ONE 12:e0183272

[53] Scheres SH (2012a) A Bayesian view on cryo-EM structure determination. J Mol Biol 415:406–418

[54] Scheres SH (2012b) RELION: implementation of a Bayesian approach to cryo-EM structure determination. J Struct Biol 180:519–530

[55] Scheres SH, Chen S (2012) Prevention of overfitting in cryo-EM structure determination. Nat Methods 9:853–854

[56] Shimoji K, Jakovljevic J, Tsuchihashi K, Umeki Y, Wan K, Kawasaki S, Talkish J, Woolford JL Jr, Mizuta K (2012) Ebp2 and Brx1 function cooperatively in 60S ribosomal subunit assembly in Saccharomyces cerevisiae. Nucleic Acids Res 40:4574–4588

[57] Sun Q, Zhu X, Qi J, An W, Lan P, Tan D, Chen R, Wang B, Zheng S, Zhang C (2017) Molecular architecture of the 90S small subunit pre-ribosome. Elife 6:e22086

[58] Talkish J, Zhang J, Jakovljevic J, Horsey EW, Woolford JL Jr (2012) Hierarchical recruitment into nascent ribosomes of assembly factors required for 27SB pre-rRNA processing in Saccharomyces cerevisiae. Nucleic Acids Res 40:8646–8661

[59] Thoms M, Thomson E, Bassler J, Gnadig M, Griesel S, Hurt E (2015) The exosome is recruited to RNA substrates through specific adaptor proteins. Cell 162:1029–1038

[60] Ulbrich C, Diepholz M, Bassler J, Kressler D, Pertschy B, Galani K, Bottcher B, Hurt E (2009) Mechanochemical removal of ribosome biogenesis factors from nascent 60S ribosomal subunits. Cell 138:911–922

[61] Vonrhein C, Blanc E, Roversi P, Bricogne G (2007) Automated structure solution with autoSHARP. Methods Mol Biol 364:215–230

[62] Wehner KA, Baserga SJ (2002) The sigma(70)-like motif: a eukaryotic RNA binding domain unique to a superfamily of proteins required for ribosome biogenesis. Mol Cell 9:329–339

[63] Weis F, Giudice E, Churcher M, Jin L, Hilcenko C, Wong CC, Traynor D, Kay RR, Warren AJ (2015) Mechanism of eIF6 release from the nascent 60S ribosomal subunit. Nat Struct Mol Biol 22:914–919

[64] Woolford JL Jr, Baserga SJ (2013) Ribosome biogenesis in the yeast Saccharomyces cerevisiae. Genetics 195:643–681

[65] Wu S, Tutuncuoglu B, Yan K, Brown H, Zhang Y, Tan D, Gamalinda M, Yuan Y, Li Z, Jakovljevic J (2016) Diverse roles of assembly factors revealed by structures of late nuclear pre-60S ribosomes. Nature 534:133–137

[66] Yang Q, Del Campo M, Lambowitz AM, Jankowsky E (2007) DEADbox proteins unwind duplexes by local strand separation. Mol Cell 28:253–263

[67] Yang B, Wu YJ, Zhu M, Fan SB, Lin J, Zhang K, Li S, Chi H, Li YX, Chen HF, Luo SK, Ding YH, Wang LH, Hao Z, Xiu LY, Chen S, Ye K, He SM, Dong MQ (2012) Identification of cross-linked peptides from complex samples. Nat Methods 9(9):904–906

[68] Zhang L, Wu C, Cai G, Chen S, Ye K (2016) Stepwise and dynamic assembly of the earliest precursors of small ribosomal subunits in yeast. Genes Dev 30:718–732

[69] Zheng S, Ye K (2014) Purification, crystallization and preliminary X-ray diffraction analysis of Imp3 in complex with an Mpp10 peptide involved in yeast ribosome biogenesis. Acta Crystallogr F Struct Biol Commun 70:918–921

[70] Zheng S, Lan P, Liu X, Ye K (2014) Interaction between ribosome assembly factors Krr1 and Faf1 is essential for formation of small ribosomal subunit in yeast. J Biol Chem 289:22692–22703

RIGHTS & PERMISSIONS

2018 The Author(s) 2018