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

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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.

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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.

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.

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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.

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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

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

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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

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