Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Cryo-EM structure of the yeast U4/U6.U5 tri-snRNP at 3.7 Å resolution

This article has been updated

Abstract

U4/U6.U5 tri-snRNP represents a substantial part of the spliceosome before activation. A cryo-electron microscopy structure of Saccharomyces cerevisiae U4/U6.U5 tri-snRNP at 3.7 Å resolution led to an essentially complete atomic model comprising 30 proteins plus U4/U6 and U5 small nuclear RNAs (snRNAs). The structure reveals striking interweaving interactions of the protein and RNA components, including extended polypeptides penetrating into subunit interfaces. The invariant ACAGAGA sequence of U6 snRNA, which base-pairs with the 5′-splice site during catalytic activation, forms a hairpin stabilized by Dib1 and Prp8 while the adjacent nucleotides interact with the exon binding loop 1 of U5 snRNA. Snu114 harbours GTP, but its putative catalytic histidine is held away from the γ-phosphate by hydrogen bonding to a tyrosine in the amino-terminal domain of Prp8. Mutation of this histidine to alanine has no detectable effect on yeast growth. The structure provides important new insights into the spliceosome activation process leading to the formation of the catalytic centre.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Three orthogonal views of a near-complete atomic model of the Saccharomyces cerevisiae U4/U6.U5 tri-snRNP.
Figure 2: Prp8 and U4/U6 and U5 snRNAs.
Figure 3: Snu114 and its interaction with guanine nucleotide, Prp8 and U5 snRNA.
Figure 4: Interactions of U4/U6 snRNAs with proteins.
Figure 5: B complex formation and activation mechanism.

Similar content being viewed by others

Accession codes

Primary accessions

Electron Microscopy Data Bank

Protein Data Bank

Data deposits

The cryo-EM maps have been deposited in the Electron Microscopy Data Bank with accession codes EMD-8006, EMD-8007, EMD-8008, EMD-8009, EMD-8010, EMD-8011, EMD-8012, EMD-8013 and EMD-8014. The coordinates of the atomic models have been deposited in the Protein Data Bank under accession codes 5GAN (overall), 5GAP (body domain), 5GAO (head domain) and 5GAM (foot domain).

Change history

  • 17 February 2016

    The Electron Microscopy Data Bank accession codes were listed in full, rather than as a range.

References

  1. Will, C. L. & Lührmann, R. Spliceosome structure and function. Cold Spring Harb. Perspect. Biol. 3, a003707 (2011)

    CAS  Google Scholar 

  2. Chen, C. H. et al. Functional and physical interactions between components of the Prp19p-associated complex. Nucleic Acids Res. 30, 1029–1037 (2002)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Fabrizio, P. et al. The evolutionarily conserved core design of the catalytic activation step of the yeast spliceosome. Mol. Cell 36, 593–608 (2009)

    Article  CAS  PubMed  Google Scholar 

  4. Ohrt, T. et al. Molecular dissection of step 2 catalysis of yeast pre-m RNA splicing investigated in a purified system. RNA 19, 902–915 (2013)

    CAS  Google Scholar 

  5. Staley, J. P. & Guthrie, C. An RNA switch at the 5′ splice site requires ATP and the DEAD box protein Prp28p. Mol. Cell 3, 55–64 (1999)

    Article  CAS  PubMed  Google Scholar 

  6. Raghunathan, P. L. & Guthrie, C. RNA unwinding in U4/U6 snRNPs requires ATP hydrolysis and the DEIH-box splicing factor Brr2. Curr. Biol. 8, 847–855 (1998)

    Article  CAS  PubMed  Google Scholar 

  7. Laggerbauer, B., Achsel, T. & Lührmann, R. The human U5-200kD DEXH-box protein unwinds U4/U6 RNA duplices in vitro. Proc. Natl Acad. Sci. USA 95, 4188–4192 (1998)

    Article  ADS  CAS  PubMed  Google Scholar 

  8. Fica, S. M. et al. RNA catalyses nuclear pre-mRNA splicing. Nature 503, 229–234 (2013)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  9. Madhani, H. D. & Guthrie, C. Dynamic RNA-RNA interactions in the spliceosome. Annu. Rev. Genet. 28, 1–26 (1994)

    Article  CAS  PubMed  Google Scholar 

  10. Toor, N., Keating, K. S., Taylor, S. D. & Pyle, A. M. Crystal structure of a self-spliced group II intron. Science 320, 77–82 (2008)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  11. Fica, S. M., Mefford, M. A., Piccirilli, J. A. & Staley, J. P. Evidence for a group II intron-like catalytic triplex in the spliceosome. Nature Struct. Mol. Biol. 21, 464–471 (2014)

    Article  CAS  Google Scholar 

  12. Newman, A. J. & Norman, C. U5 snRNA interacts with exon sequences at 5′ and 3′ splice sites. Cell 68, 743–754 (1992)

    Article  CAS  PubMed  Google Scholar 

  13. Sontheimer, E. J. & Steitz, J. A. The U5 and U6 small nuclear RNAs as active site components of the spliceosome. Science 262, 1989–1996 (1993)

    Article  ADS  CAS  PubMed  Google Scholar 

  14. Stark, H. & Lührmann, R. Cryo-electron microscopy of spliceosomal components. Annu. Rev. Biophys. Biomol. Struct. 35, 435–457 (2006)

    Article  CAS  PubMed  Google Scholar 

  15. Bai, X. C., McMullan, G. & Scheres, S. H. How cryo-EM is revolutionizing structural biology. Trends Biochem. Sci. 40, 49–57 (2015)

    Article  CAS  PubMed  Google Scholar 

  16. Nguyen, T. H. et al. The architecture of the spliceosomal U4/U6.U5 tri-snRNP. Nature 523, 47–52 (2015)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  17. Ohi, M. D., Ren, L., Wall, J. S., Gould, K. L. & Walz, T. Structural characterization of the fission yeast U5.U2/U6 spliceosome complex. Proc. Natl Acad. Sci. USA 104, 3195–3200 (2007)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  18. Chen, W. et al. Endogenous U2·U5·U6 snRNA complexes in S. pombe are intron lariat spliceosomes. RNA 20, 308–320 (2014)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Yan, C. et al. Structure of a yeast spliceosome at 3.6-angstrom resolution. Science 349, 1182–1191 (2015)

    Article  ADS  CAS  PubMed  Google Scholar 

  20. Hang, J., Wan, R., Yan, C. & Shi, Y. Structural basis of pre-mRNA splicing. Science 349, 1191–1198 (2015)

    Article  ADS  CAS  PubMed  Google Scholar 

  21. Stevens, S. W. et al. Biochemical and genetic analyses of the U5, U6, and U4/U6 x U5 small nuclear ribonucleoproteins from Saccharomyces cerevisiae. RNA 7, 1543–1553 (2001)

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Gottschalk, A. et al. Identification by mass spectrometry and functional analysis of novel proteins of the yeast [U4/U6.U5] tri-snRNP. EMBO J. 18, 4535–4548 (1999)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Bai, X.-C., Rajendra, E., Yang, G., Shi, Y. & Scheres, S. H. W. Sampling the conformational space of the catalytic subunit of human γ-secretase. eLife 4, e11182 (2015)

    Article  PubMed  PubMed Central  Google Scholar 

  24. Galej, W. P., Oubridge, C., Newman, A. J. & Nagai, K. Crystal structure of Prp8 reveals active site cavity of the spliceosome. Nature 493, 638–643 (2013)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  25. Reuter, K., Nottrott, S., Fabrizio, P., Lührmann, R. & Ficner, R. Identification, characterization and crystal structure analysis of the human spliceosomal U5 snRNP-specific 15 kD protein. J. Mol. Biol. 294, 515–525 (1999)

    Article  CAS  PubMed  Google Scholar 

  26. Fabrizio, P., Laggerbauer, B., Lauber, J., Lane, W. S. & Lührmann, R. An evolutionarily conserved U5 snRNP-specific protein is a GTP-binding factor closely related to the ribosomal translocase EF-2. EMBO J. 16, 4092–4106 (1997)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Jørgensen, R. et al. Two crystal structures demonstrate large conformational changes in the eukaryotic ribosomal translocase. Nature Struct. Biol. 10, 379–385 (2003)

    Article  PubMed  CAS  Google Scholar 

  28. Wittinghofer, A. & Vetter, I. R. Structure-function relationships of the G domain, a canonical switch motif. Annu. Rev. Biochem. 80, 943–971 (2011)

    Article  CAS  PubMed  Google Scholar 

  29. Tourigny, D. S., Fernández, I. S., Kelley, A. C. & Ramakrishnan, V. Elongation factor G bound to the ribosome in an intermediate state of translocation. Science 340, 1235490 (2013)

    Article  PubMed  CAS  Google Scholar 

  30. Lin, J., Gagnon, M. G., Bulkley, D. & Steitz, T. A. Conformational changes of elongation factor G on the ribosome during tRNA translocation. Cell 160, 219–227 (2015)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Brenner, T. J. & Guthrie, C. Genetic analysis reveals a role for the C terminus of the Saccharomyces cerevisiae GTPase Snu114 during spliceosome activation. Genetics 170, 1063–1080 (2005)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Maracci, C., Peske, F., Dannies, E., Pohl, C. & Rodnina, M. V. Ribosome-induced tuning of GTP hydrolysis by a translational GTPase. Proc. Natl Acad. Sci. USA 111, 14418–14423 (2014)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  33. Small, E. C., Leggett, S. R., Winans, A. A. & Staley, J. P. The EF-G-like GTPase Snu114p regulates spliceosome dynamics mediated by Brr2p, a DExD/H box ATPase. Mol. Cell 23, 389–399 (2006)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Bartels, C., Urlaub, H., Lührmann, R. & Fabrizio, P. Mutagenesis suggests several roles of Snu114p in pre-mRNA splicing. J. Biol. Chem. 278, 28324–28334 (2003)

    Article  CAS  PubMed  Google Scholar 

  35. Brenner, T. J. & Guthrie, C. Assembly of Snu114 into U5 snRNP requires Prp8 and a functional GTPase domain. RNA 12, 862–871 (2006)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Liu, S. et al. Binding of the human Prp31 Nop domain to a composite RNA-protein platform in U4 snRNP. Science 316, 115–120 (2007)

    Article  ADS  CAS  PubMed  Google Scholar 

  37. Ayadi, L. et al. Functional and structural characterization of the Prp3 binding domain of the yeast Prp4 splicing factor. J. Mol. Biol. 284, 673–687 (1998)

    Article  CAS  PubMed  Google Scholar 

  38. Liu, S. et al. A composite double-/single-stranded RNA-binding region in protein Prp3 supports tri-snRNP stability and splicing. eLife 4, e07320 (2015)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  39. Nottrott, S., Urlaub, H. & Lührmann, R. Hierarchical, clustered protein interactions with U4/U6 snRNA: a biochemical role for U4/U6 proteins. EMBO J. 21, 5527–5538 (2002)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Makarov, E. M., Makarova, O. V., Achsel, T. & Lührmann, R. The human homologue of the yeast splicing factor prp6p contains multiple TPR elements and is stably associated with the U5 snRNP via protein-protein interactions. J. Mol. Biol. 298, 567–575 (2000)

    Article  CAS  PubMed  Google Scholar 

  41. Galisson, F. & Legrain, P. The biochemical defects of prp4-1 and prp6-1 yeast splicing mutants reveal that the PRP6 protein is required for the accumulation of the [U4/U6.U5] tri-snRNP. Nucleic Acids Res. 21, 1555–1562 (1993)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Nguyen, T. H. D. et al. Structural basis of Brr2-Prp8 interactions and implications for U5 snRNP biogenesis and the spliceosome active site. Structure 21, 910–919 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Hahn, D., Kudla, G., Tollervey, D. & Beggs, J. D. Brr2p-mediated conformational rearrangements in the spliceosome during activation and substrate repositioning. Genes Dev. 26, 2408–2421 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. van Nues, R. W. & Beggs, J. D. Functional contacts with a range of splicing proteins suggest a central role for Brr2p in the dynamic control of the order of events in spliceosomes of Saccharomyces cerevisiae. Genetics 157, 1451–1467 (2001)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Wolf, E. et al. Exon, intron and splice site locations in the spliceosomal B complex. EMBO J. 28, 2283–2292 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Boehringer, D. et al. Three-dimensional structure of a pre-catalytic human spliceosomal complex B. Nature Struct. Mol. Biol. 11, 463–468 (2004)

    Article  CAS  Google Scholar 

  47. De, I. et al. The RNA helicase Aquarius exhibits structural adaptations mediating its recruitment to spliceosomes. Nature Struct. Mol. Biol. 22, 138–144 (2015)

    Article  CAS  Google Scholar 

  48. Li, Z. & Brow, D. A. A spontaneous duplication in U6 spliceosomal RNA uncouples the early and late functions of the ACAGA element in vivo. RNA 2, 879–894 (1996)

    CAS  PubMed  Google Scholar 

  49. Kuhn, A. N. & Brow, D. A. Suppressors of a cold-sensitive mutation in yeast U4 RNA define five domains in the splicing factor Prp8 that influence spliceosome activation. Genetics 155, 1667–1682 (2000)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Chan, S. P. & Cheng, S. C. The Prp19-associated complex is required for specifying interactions of U5 and U6 with pre-mRNA during spliceosome activation. J. Biol. Chem. 280, 31190–31199 (2005)

    Article  CAS  PubMed  Google Scholar 

  51. Li, X. et al. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nature Methods 10, 584–590 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Mindell, J. A. & Grigorieff, N. Accurate determination of local defocus and specimen tilt in electron microscopy. J. Struct. Biol. 142, 334–347 (2003)

    Article  PubMed  Google Scholar 

  53. Scheres, S. H. A Bayesian view on cryo-EM structure determination. J. Mol. Biol. 415, 406–418 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Scheres, S. H. Beam-induced motion correction for sub-megadalton cryo-EM particles. eLife 3, e03665 (2014)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  55. Kucukelbir, A., Sigworth, F. J. & Tagare, H. D. Quantifying the local resolution of cryo-EM density maps. Nature Methods 11, 63–65 (2014)

    Article  CAS  PubMed  Google Scholar 

  56. Scheres, S. H. & Chen, S. Prevention of overfitting in cryo-EM structure determination. Nature Methods 9, 853–854 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Chen, S. et al. High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy. Ultramicroscopy 135, 24–35 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Murshudov, G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D 67, 355–367 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Nicholls, R. A., Fischer, M., McNicholas, S. & Murshudov, G. N. Conformation-independent structural comparison of macromolecules with ProSMART. Acta Crystallogr. D 70, 2487–2499 (2014)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Brown, A. et al. Tools for macromolecular model building and refinement into electron cryo-microscopy reconstructions. Acta Crystallogr. D 71, 136–153 (2015)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Scheres, S. H. W., Núñez-Ramírez, R., Sorzano, C. O. S., Carazo, J. M. & Marabini, R. Image processing for electron microscopy single-particle analysis using XMIPP. Nature Protocols 3, 977–990 (2008)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Goddard, T. D., Huang, C. C. & Ferrin, T. E. Visualizing density maps with UCSF Chimera. J. Struct. Biol. 157, 281–287 (2007)

    Article  CAS  PubMed  Google Scholar 

  64. Kunkel, T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc. Natl Acad. Sci. USA 82, 488–492 (1985)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  65. Turner, I. A., Norman, C. M., Churcher, M. J. & Newman, A. J. Dissection of Prp8 protein defines multiple interactions with crucial RNA sequences in the catalytic core of the spliceosome. RNA 12, 375–386 (2006)

    CAS  PubMed  Google Scholar 

  66. Frazer, L. N., Lovell, S. C. & O’Keefe, R. T. Analysis of synthetic lethality reveals genetic interactions between the GTPase Snu114p and snRNAs in the catalytic core of the Saccharomyces cerevisiae spliceosome. Genetics 183, 497–515 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank C. Savva, S. Chen, G. McMullan, J. Grimmett and T. Darling for running the electron microscopy and computing facilities, A. Brown, P. Emsley, G. Murshudov for advice and help with model building and refinement, R. O’Keefe for the ∆Snu114 yeast strain, the members of the spliceosome group for help and advice throughout the project and R. Leiro for help with data processing. We thank S. Fica for critical reading of the manuscript and J. Löwe, V. Ramakrishnan, and R. Henderson for their continuing support and encouragements. The project was supported by the Medical Research Council (MC_U105184330 to K.N. and MC_UP_A025_1013 to S.H.W.S.).

Author information

Authors and Affiliations

Authors

Contributions

T.H.D.N. purified yeast tri-snRNP and prepared EM grids, T.H.D.N., W.P.G. and X.-C.B collected all EM images. T.H.D.N. processed data and calculated the maps with the help of X.-C.B and S.H.W.S.; T.H.D.N., W.P.G. and C.O. built a model into the map and refined the structure. T.H.D.N. performed mutagenesis experiments with the help of A.J.N.; T.H.D.N. and W.P.G. prepared all illustrations. K.N. initiated and orchestrated the project. T.H.D.N., W.P.G. and K.N. analysed the structure and wrote the paper with invaluable contributions from all other authors.

Corresponding authors

Correspondence to Thi Hoang Duong Nguyen, Wojciech P. Galej or Kiyoshi Nagai.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Image processing procedures.

a, Representative micrograph. b, Representative 2D class averages obtained from reference-free 2D classification. c, Classification and refinement procedures used in this study.

Extended Data Figure 2 Local and overall resolutions of tri-snRNP maps.

Local resolution estimation by Resmap55 of a, the overall 3.7 Å map and b, maps of the head, body and foot domains obtained from masked refinements with signal subtraction23. c, Gold-standard FSC curves for the overall map and the maps of the head, body and foot domains obtained from masked refinements. Their resolutions are estimated at FSC = 0.143. dg, FSC curves of model versus map and cross-validation of model refinement by half-maps for the body, foot, head and overall maps, respectively. The red curves show FSC between the atomic model and the half-map it was refined against (half1) and the blue curves show FSC between the atomic model and the other half-map (half2) it was not refined against. The black curves show FSC between the atomic model and the sum map which the model was refined against.

Extended Data Figure 3 Representative EM density for different components of the map.

a, Snu114 in the Foot domain with a bound GTP (magenta). The inset shows the GTP-binding pocket. b, Brr2 in the head domain with a bound single-stranded region of U4 snRNA. The inset shows the density in the RNA binding tunnel. c, Density for Prp8 large and RNase-like domains. The inset shows the density in the core of Prp8. df, Prp3, Prp31 and Prp6 densities, respectively, with extended polypeptides.

Extended Data Figure 4 Secondary structure of the snRNAs in tri-snRNP.

a, U4/U6 snRNA; c, U5 snRNA. The coloured nucleotides with red, green and blue background were built de novo into our EM density. The region near the ACAGAGA sequence of U6 snRNA forms a stem-loop that was not predicted previously. b, d, Representative EM density for U4/U6 snRNA duplex and U5 snRNA, respectively.

Extended Data Figure 5 Interactions of Snu114 with guanine nucleotides and the N-terminal domain of Prp8 in the S. cerevisiae U4/U6.U5 tri-snRNP and S. pombe ILS complexes.

a, Conformation of the Snu114(Cwf10)-bound GDP refined in the S. pombe ILS spliceosomal complex19,20 (red, PDB 3JB9), was overlaid on GDPs found in other guanine-nucleotide binding proteins (grey, PDB coordinates: 1DAR, 2E1R, 2WRI, 1Z0I, 5CA8, 1XTQ, 4YLG, 1SF8, 5BXQ). b, Guanine nucleotide refined as GDP in Snu114 of the S. cerevisiae U4/U6.U5 tri-snRNP (blue) is overlaid on GDPs found in the PDB coordinates as in a. c, Conformation of guanine nucleotide refined as GTP in Snu114 of the S. cerevisiae U4/U6.U5 tri-snRNP (blue) agrees well with GTP or GTP analogues in other guanine-nucleotide binding proteins (PDB code: 2BV3, 2DY1, 2J7K, 4YW9, 1ASO, 1LF0 (grey)). d, Superposition of the active site of Snu114-GTP and Cwf10-GDP. e, Superposition of the GDP-bound EF-G (2WRI), GMP-PCP bound EF-G (4JUW) and Snu114 (S. cerevisiae tri-snRNP) active sites. His218 (His87 in EF-G) positions water molecule crucial for GTP hydrolysis. f, Comparison of Prp8N-term domain, Snu114 and U5 snRNA in the S. cerevisiae U4/U6.U5 complex and S. pombe ILS complex. g, Growth of serial dilutions of yeast strains carrying wild-type Snu114, His218Arg or His218Ala Snu114 mutants at different temperatures. Cells were spotted on YPD plates and grown at 14 °C for 10 days, 30 °C and 37 °C for 2 days. h, Growth of serial dilutions of yeast strains carrying wild-type Prp8, Tyr403Phe and Tyr403Ala mutants. Cells were spotted on YPD plates and grown at 14 °C for 9 days, 30 °C for 3 days. This yeast strain does not survive at 37 °C and thus is not shown.

Extended Data Figure 6 Conformational flexibility of tri-snRNP observed by classification.

a, Different conformations of the arm domain demonstrated by the unsharpened maps of the three major classes (purple, magenta and red) obtained from masked classification of the arm domain alone followed by masked refinement with the body and arm domains. The body domain was included in the refinement because the arm domain is too small for accurate alignments. b, The sharpened map of one of the three classes with Prp3 and LSm models shown. In the improved domain maps for the arm domain, extra density for the N-terminal helix of Prp3 could be observed to extend to the LSm proteins. c, The sharpened map of the tri-snRNP and the locations of Snu66 and Prp8. d, The open and closed conformations of the head and foot domains of the tri-snRNP observed by global classification. The unsharpened maps for the two major classes obtained from global classification with finer angular sampling (1.8°) followed by 3D auto-refinement are shown. The open and closed states are indicated. e, Superposition of the unsharpened maps of the open (grey) and closed (yellow) states shown in d. The arrows indicate the rotations of the head and foot domains.

Extended Data Figure 7 Brr2 helicase and its U4/U6 snRNA substrate.

a, Domain structure of Brr2 helicase comprising the N-terminal domain and two helicase cassettes. Individual domains of N-terminal helicase cassette (NHC) are colour-coded. b, Extensive interactions of Brr2 with U4/U6 snRNA and Prp3. The single-stranded region of U4 snRNA extending from stem I enters the active site near the β-finger (red). c, 3′ stem of U4 snRNA interacts with the HLH domain of NHC. d, The N-terminal domain (NTD) of Brr2 interacts with a long helix of Prp3 and inserts a loop into U4/U6 Stem II. e, Snu66 has a long extended region that wraps around both helicase cassettes of Brr2.

Extended Data Table 1 Summary of model building of tri-snRNP components
Extended Data Table 2 Refinement, model statistics and structure/map depositions

Supplementary information

Supplementary Data

This zipped file contains the pymol session file of the PDB coordinate. (ZIP 4054 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Nguyen, T., Galej, W., Bai, Xc. et al. Cryo-EM structure of the yeast U4/U6.U5 tri-snRNP at 3.7 Å resolution. Nature 530, 298–302 (2016). https://doi.org/10.1038/nature16940

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature16940

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing