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Structure of a spliceosome remodelled for exon ligation


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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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.

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Authors and Affiliations



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).

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