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Structure of a spliceosome remodelled for exon ligation

Abstract

The spliceosome excises introns from pre-mRNAs in two sequential transesterifications—branching and exon ligation1—catalysed at a single catalytic metal site in U6 small nuclear RNA (snRNA)2,3. Recently reported structures of the spliceosomal C complex4,5 with the cleaved 5′ exon and lariat–3′-exon bound to the catalytic centre revealed that branching-specific factors such as Cwc25 lock the branch helix into position for nucleophilic attack of the branch adenosine at the 5′ splice site. Furthermore, the ATPase Prp16 is positioned to bind and translocate the intron downstream of the branch point to destabilize branching-specific factors and release the branch helix from the active site4. Here we present, at 3.8 Å resolution, the cryo-electron microscopy structure of a Saccharomyces cerevisiae spliceosome stalled after Prp16-mediated remodelling but before exon ligation. While the U6 snRNA catalytic core remains firmly held in the active site cavity of Prp8 by proteins common to both steps, the branch helix has rotated by 75° compared to the C complex and is stabilized in a new position by Prp17, Cef1 and the reoriented Prp8 RNase H-like domain. This rotation of the branch helix removes the branch adenosine from the catalytic core, creates a space for 3′ exon docking, and restructures the pairing of the 5′ splice site with the U6 snRNA ACAGAGA region. Slu7 and Prp18, which promote exon ligation, bind together to the Prp8 RNase H-like domain. The ATPase Prp22, bound to Prp8 in place of Prp16, could interact with the 3′ exon, suggesting a possible basis for mRNA release after exon ligation6,7. Together with the structure of the C complex4, our structure of the C* complex reveals the two major conformations of the spliceosome during the catalytic stages of splicing.

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Figure 1: Subunit organization of the C* spliceosome.
Figure 2: Architecture of the RNA catalytic core in the C* complex.
Figure 3: Proteins stabilize the repositioned branch helix.
Figure 4: Prp22 and ATP-mediated transitions at the catalytic stage of splicing.

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References

  1. Wahl, M. C., Will, C. L. & Lührmann, R. The spliceosome: design principles of a dynamic RNP machine. Cell 136, 701–718 (2009)

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  3. Madhani, H. D. & Guthrie, C. A novel base-pairing interaction between U2 and U6 snRNAs suggests a mechanism for the catalytic activation of the spliceosome. Cell 71, 803–817 (1992)

    Article  CAS  Google Scholar 

  4. Galej, W. P. et al. Cryo-EM structure of the spliceosome immediately after branching. Nature 537, 197–201 (2016)

    Article  ADS  CAS  Google Scholar 

  5. Wan, R., Yan, C., Bai, R., Huang, G. & Shi, Y. Structure of a yeast catalytic step I spliceosome at 3.4 Å resolution. Science 353, 895–904 (2016)

    Article  ADS  CAS  Google Scholar 

  6. Schwer, B. A conformational rearrangement in the spliceosome sets the stage for Prp22-dependent mRNA release. Mol. Cell 30, 743–754 (2008)

    Article  CAS  Google Scholar 

  7. Semlow, D. R., Blanco, M. R., Walter, N. G. & Staley, J. P. Spliceosomal DEAH-box ATPases remodel pre-mRNA to activate alternative splice sites. Cell 164, 985–998 (2016)

    Article  CAS  Google Scholar 

  8. Schwer, B. & Guthrie, C. PRP16 is an RNA-dependent ATPase that interacts transiently with the spliceosome. Nature 349, 494–499 (1991)

    Article  ADS  CAS  Google Scholar 

  9. Tseng, C. K., Liu, H. L. & Cheng, S. C. DEAH-box ATPase Prp16 has dual roles in remodeling of the spliceosome in catalytic steps. RNA 17, 145–154 (2011)

    Article  CAS  Google Scholar 

  10. James, S.-A., Turner, W. & Schwer, B. How Slu7 and Prp18 cooperate in the second step of yeast pre-mRNA splicing. RNA 8, 1068–1077 (2002)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  12. Brys, A. & Schwer, B. Requirement for SLU7 in yeast pre-mRNA splicing is dictated by the distance between the branchpoint and the 3′ splice site. RNA 2, 707–717 (1996)

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Chua, K. & Reed, R. The RNA splicing factor hSlu7 is required for correct 3′ splice-site choice. Nature 402, 207–210 (1999)

    Article  ADS  CAS  Google Scholar 

  14. Moore, M. J. & Sharp, P. A. Site-specific modification of pre-mRNA: the 2′-hydroxyl groups at the splice sites. Science 256, 992–997 (1992)

    Article  ADS  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  17. 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  Google Scholar 

  18. Siatecka, M., Reyes, J. L. & Konarska, M. M. Functional interactions of Prp8 with both splice sites at the spliceosomal catalytic center. Genes Dev. 13, 1983–1993 (1999)

    Article  CAS  Google Scholar 

  19. Collins, C. A. & Guthrie, C. Genetic interactions between the 5′ and 3′ splice site consensus sequences and U6 snRNA during the second catalytic step of pre-mRNA splicing. RNA 7, 1845–1854 (2001)

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Jacquier, A. & Michel, F. Base-pairing interactions involving the 5′ and 3′-terminal nucleotides of group II self-splicing introns. J. Mol. Biol. 213, 437–447 (1990)

    Article  CAS  Google Scholar 

  21. Konarska, M. M., Vilardell, J. & Query, C. C. Repositioning of the reaction intermediate within the catalytic center of the spliceosome. Mol. Cell 21, 543–553 (2006)

    Article  CAS  Google Scholar 

  22. Villa, T. & Guthrie, C. The Isy1p component of the NineTeen complex interacts with the ATPase Prp16p to regulate the fidelity of pre-mRNA splicing. Genes Dev. 19, 1894–1904 (2005)

    Article  CAS  Google Scholar 

  23. Jiang, J., Horowitz, D. S. & Xu, R. M. Crystal structure of the functional domain of the splicing factor Prp18. Proc. Natl Acad. Sci. USA 97, 3022–3027 (2000)

    Article  ADS  CAS  Google Scholar 

  24. Zhang, X. & Schwer, B. Functional and physical interaction between the yeast splicing factors Slu7 and Prp18. Nucleic Acids Res. 25, 2146–2152 (1997)

    Article  CAS  Google Scholar 

  25. Luukkonen, B. G. & Séraphin, B. The role of branchpoint-3′ splice site spacing and interaction between intron terminal nucleotides in 3′ splice site selection in Saccharomyces cerevisiae. EMBO J. 16, 779–792 (1997)

    Article  CAS  Google Scholar 

  26. Steitz, T. A. & Steitz, J. A. A general two-metal-ion mechanism for catalytic RNA. Proc. Natl Acad. Sci. USA 90, 6498–6502 (1993)

    Article  ADS  CAS  Google Scholar 

  27. Frank, D. & Guthrie, C. An essential splicing factor, SLU7, mediates 3′ splice site choice in yeast. Genes Dev. 6, 2112–2124 (1992)

    Article  CAS  Google Scholar 

  28. Schwer, B. & Gross, C. H. Prp22, a DExH-box RNA helicase, plays two distinct roles in yeast pre-mRNA splicing. EMBO J. 17, 2086–2094 (1998)

    Article  CAS  Google Scholar 

  29. He, Y., Andersen, G. R. & Nielsen, K. H. Structural basis for the function of DEAH helicases. EMBO Rep. 11, 180–186 (2010)

    Article  CAS  Google Scholar 

  30. Hilliker, A. K., Mefford, M. A. & Staley, J. P. U2 toggles iteratively between the stem IIa and stem IIc conformations to promote pre-mRNA splicing. Genes Dev. 21, 821–834 (2007)

    Article  CAS  Google Scholar 

  31. Abelson, J., Hadjivassiliou, H. & Guthrie, C. Preparation of fluorescent pre-mRNA substrates for an smFRET study of pre-mRNA splicing in yeast. Methods Enzymol. 472, 31–40 (2010)

    Article  CAS  Google Scholar 

  32. Zhou, Z., Licklider, L. J., Gygi, S. P. & Reed, R. Comprehensive proteomic analysis of the human spliceosome. Nature 419, 182–185 (2002)

    Article  ADS  CAS  Google Scholar 

  33. Umen, J. G. & Guthrie, C. A novel role for a U5 snRNP protein in 3′ splice site selection. Genes Dev. 9, 855–868 (1995)

    Article  CAS  Google Scholar 

  34. Lin, R. J., Newman, A. J., Cheng, S. C. & Abelson, J. Yeast mRNA splicing in vitro. J. Biol. Chem. 260, 14780–14792 (1985)

    CAS  PubMed  Google Scholar 

  35. Nguyen, T. H. D. et al. Cryo-EM structure of the yeast U4/U6.U5 tri-snRNP at 3.7 Å resolution. Nature 530, 298–302 (2016)

    Article  ADS  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  37. Rohou, A. & Grigorieff, N. CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015)

    Article  Google Scholar 

  38. Scheres, S. H. W. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012)

    Article  CAS  Google Scholar 

  39. Scheres, S. H. Processing of structurally heterogeneous cryo-EM data in RELION. Methods Enzymol. 579, 125–157 (2016)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  41. 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  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  43. Biasini, M. et al. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res. 42, W252–W258 (2014)

    Article  CAS  Google Scholar 

  44. Kozlowski, L. P. & Bujnicki, J. M. MetaDisorder: a meta-server for the prediction of intrinsic disorder in proteins. BMC Bioinformatics 13, 111 (2012)

    Article  ADS  Google Scholar 

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

    Article  CAS  Google Scholar 

  46. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997)

    Article  CAS  Google Scholar 

  47. 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  Google Scholar 

  48. 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  Google Scholar 

  49. Zwart, P. H. et al. Automated structure solution with the PHENIX suite. Methods Mol. Biol. 426, 419–435 (2008)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  51. Yan, C., Wan, R., Bai, R., Huang, G. & Shi, Y. Structure of a yeast activated spliceosome at 3.5 Å resolution. Science 353, 904–911 (2016)

    Article  ADS  CAS  Google Scholar 

  52. Marcia, M. & Pyle, A. M. Visualizing group II intron catalysis through the stages of splicing. Cell 151, 497–507 (2012)

    Article  CAS  Google Scholar 

  53. Robart, A. R., Chan, R. T., Peters, J. K., Rajashankar, K. R. & Toor, N. Crystal structure of a eukaryotic group II intron lariat. Nature 514, 193–197 (2014)

    Article  ADS  CAS  Google Scholar 

  54. Aronova, A., Bacíková, D., Crotti, L. B., Horowitz, D. S. & Schwer, B. Functional interactions between Prp8, Prp18, Slu7, and U5 snRNA during the second step of pre-mRNA splicing. RNA 13, 1437–1444 (2007)

    Article  CAS  Google Scholar 

  55. Davis, I. W., Murray, L. W., Richardson, J. S. & Richardson, D. C. MOLPROBITY: structure validation and all-atom contact analysis for nucleic acids and their complexes. Nucleic Acids Res. 32, W615–W619 (2004)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank S. Scheres for his help and advice on data collection and processing; C. Savva, S. Chen, K. R. Vinothkumar, G. McMullan, J. Grimmett and T. Darling for smooth running of the EM and computing facilities; the staff at Diamond Light Source (DLS) for help with data collection; the mass spectrometry facility for help with protein identification, P. Emsley and G. Murshudov for help and advice with model building and refinement; the members of the spliceosome group for help and advice throughout the project. We thank J. Löwe, V. Ramakrishnan, D. Barford and R. Henderson for their continuing support, C. Plaschka, P. C. Lin and L. Strittmatter for critical reading of the manuscript and J. Vilardell for a generous gift of reagent. The project was supported by the Medical Research Council (MC_U105184330) and European Research Council Advanced Grant (693087 - SPLICE3D). S.M.F. was supported by EMBO and Marie Skłodowska-Curie fellowships, M.E.W was supported by a Rutherford Memorial Cambridge Scholarship.

Author information

Authors and Affiliations

Authors

Contributions

S.M.F. and W.P.G. established the method of C* complex preparation. S.M.F. prepared the EM sample and grids, S.M.F., W.P.G., M.E.W. and X.C.B. collected and processed EM data, C.O., S.M.F., W.P.G. and M.E.W. carried out model building and C.O. refined and finalized the PDB file. S.M.F., C.O., W.P.G., M.E.W. and K.N. analysed the structure. A.J.N. prepared the substrate and contributed to the project through his knowledge and experience on yeast splicing. Manuscript was initially written by S.M.F. and K.N. and finalized with input from all authors. K.N. initiated and coordinated the spliceosome project.

Corresponding authors

Correspondence to Sebastian M. Fica or Kiyoshi Nagai.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Purification and cryo-EM imaging of the C* spliceosome.

a, Protein composition of the purified C* complex. Note that Prp16 is strongly de-enriched compared to Prp22, consistent with the majority of the purified complexes being in a post-Prp16 conformation, as Prp16 dissociates upon ATP hydrolysis8. b, The purified C* complex contains mostly lariat intermediates and catalyses exon ligation with low efficiency when incubated in the presence of Mg2+. The identity of the major species, inferred by size and migration pattern, is indicated by the cartoon on the left. c, Representative electron micrograph of the C* complex sample collected at 3 μm defocus. d, Representative 2D class averages of the C* complex obtained in RELION39. e, Image of a highly abundant C* complex 2D class average illustrating the major domains of the complex.

Extended Data Figure 2 Data processing workflow.

a, Method used to obtain an initial model of the C* complex for 3D classification in RELION39. b, Scheme for 3D classification and refinement. The mask used to obtain the overall 3.8 Å map excluded the Clf1 and Syf1 arch regions as well as the Prp22 and U2 snRNP regions. Note that focused classification was performed without signal subtraction. All nominal resolutions reported were obtained during post-processing in RELION39.

Extended Data Figure 3 Angular distribution and FSC curves for the C* reconstructions.

a, Angular distribution for the 3.8 Å map of the core region. Note the presence of several orthogonal views. b, Overall reconstructions obtained after classification with masks on the core (grey), the core with Prp22 (magenta), and the core with the U2 snRNP (green). The three maps were superposed and aligned on the core using Chimera50. c, Gold-standard FSC curves for the three maps shown in b. d, Local resolution for the core map, calculated using RELION 2.0 (ref. 39).

Extended Data Figure 4 Fit of the model built into the C* map.

a, Experimental density for Prp22 and fitting of the model into the density. bd, Fitting of the model into the experimental density for key regions of the C* map. e, Fourier shell correlation between model and the map and cross-validation of the model fitting. The original atom positions have been randomly displaced up to 0.5 Å and refined with restraints against the half1 map only. FSC was calculated for the two half maps. Excellent correlation up to the high resolution between the model and the half2 map (which was not used in refinement) cross-validates the model for overfitting.

Extended Data Figure 5 U2 snRNP rearrangement between C and C* complexes and repositioning of the Prp8 RH domain during splicing.

a, b, Movement of the U2 snRNP domain between the C complex (a) and the C* complex (b). Note that U2 stem IIa switches from an interaction with Prp17 in C complex to a position adjacent to the U2 Sm ring in C*; the binding of U2 stem IIb by Ecm2/Cwc2 is disrupted and Prp17 changes its binding surface on Ecm2/Cwc2. In a, Brr2 was omitted for clarity. c, Prp8 RH domain conformation in the Bact complex (PDB 5GM6; ref. 51). Note how Hsh155 sequesters the branch helix away from the RH domain, which projects its β-hairpin into solvent and is stabilized by the Prp8 Jab1/MPN domain and Brr2 (not shown). d, e, RH conformation in the C (d) and C* (e) complexes. Note that the RH domain undergoes a dramatic inward rotation towards the body of the complex and is stabilized in alternative conformations by factors specific for branching (C) or exon ligation (C*). f, RH conformation in the Schizosaccharomyces pombe ILS. Note that the de-branching specific factor Cwf19 is now wedged between the RH domain and the branch helix. All structures were aligned on the Prp8 endonuclease domain (Prp8EN), shown in grey; complex-specific factors are coloured in magenta shades; Prp8RH, Prp8 RH domain.

Extended Data Figure 6 Metals in the RNA core of the C* complex.

a, b, Structure (a) and schematic representation (b) of the active site of a group IIC intron trapped in the post-catalytic state in the presence of Mg2+ and K+ (PDB 4FAR, ref. 52). The 5′-exon 3′ hydroxyl interacts with M1, while a water molecule bridges the two catalytic metals. Two additional non-catalytic Mg2+ and two K+ close to the active site are also shown. ce, Structure of the RNA at the active site of the spliceosomal C* complex, with putative metal binding (c), schematic of catalytic metal binding (M1 and M2) (d), and comparison of the putative metal binding model with the EM density (e). Note conservation of the metal binding residues compared to the group II intron and proximity of the cleaved G(−1) 3′ hydroxyl to M1. Besides the two catalytic Mg2+, additional divalent and monovalent metals were observed in the group IIB structure53. Density observed at analogous position in C* complex may be attributable to a Mg2+ (M3) and two K+ (M4 and M5). f, Proposed interactions between U6 snRNA and the two catalytic Mg2+ during the transition state for exon ligation, as inferred from biochemistry (ref. 2). g, h, Structure (g) and schematic (h) of the RNA core of a group IIB intron in a post-catalytic configuration, following both branching and exon ligation (PDB 4R0D, ref. 53). Residues that position the catalytic metals are shown in magenta.

Extended Data Figure 7 Structure and interactions of Slu7 and Prp18.

a, b, Overall arrangement of modelled regions for Slu7 and Prp18 in cartoon (a) and surface (b) representation. The dashed lines in a represent inferred chain continuity based on regions of weak density. Note how Slu7 latches the Prp8 RH domain onto the Prp8 endonuclease (Prp8EN) and N-terminal domains (Prp8N). c, Interaction between Slu7 and Prp18. The Slu7 helix was modelled on the basis of secondary structure predictions and previously reported genetic interactions10,24. d, Interactions between Slu7 and Prp8. The R1753A allele impairs exon ligation and interacts genetically with Slu7 (ref. 54). IP6, inositol hexakisphosphate. e, f, Exchange of Cwc25 in the C complex (e) for Slu7 in the C* complex (f) on Prp8. Note that Slu7 binding is stabilized in C* by an α-helix of Prp8 (residues 2090–2110) that would clash with Cwc25 in the C complex; indeed, this helix is not visible in the C complex and only becomes ordered in C*.

Extended Data Figure 8 Model for the role of Prp18/Slu7 in 3′ exon docking.

a, Putative model for insertion of Prp18 conserved region (188–222) into the catalytic core of the C* complex. Orthogonal views of Prp8 cradling the catalytic RNA core are shown. Note accessible channel facing the location of Prp18. The conserved region, which is missing from the crystal structure and is not visible in our cryo-EM map, is shown as a dotted line. b, Distances between key elements involved in exon ligation visible in our C* map. The possible path of the intron between the last visible residue of the intron (A73) and the 5′ exon G(−1) is shown as a dotted black line. Note that six nucleotides (nt) of A-form RNA would be sufficient to reach the 5′ exon. Roughly 28 amino acids (aa) of fully extended protein would be sufficient to reach the 5′ exon; the Prp18 conserved region is 34 amino acids in length. c, Steps in pre-mRNA splicing. BrA, branch point adenosine. d, Cartoon model for 3′ splice site docking and exon ligation. For pre-mRNAs with a short distance from branch point (BP) to 3′ splice site, Prp16 action could be sufficient to allow docking of the 3′ splice site. For longer distances from branch point to 3′ splice site, Prp18 and Slu7 could become indispensable to guide the 3′ splice site to the active site. Indeed, for the UBC4 intron, Prp16 activity is not sufficient for 3′ splice site docking, which requires Slu7/Prp18 (ref. 7).

Extended Data Table 1 Cryo-EM data collection and refinement statistics
Extended Data Table 2 Summary of components modelled into the C* complex map

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Fica, S., Oubridge, C., Galej, W. et al. Structure of a spliceosome remodelled for exon ligation. Nature 542, 377–380 (2017). https://doi.org/10.1038/nature21078

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