Abstract
Precursor mRNA (pre-mRNA) splicing proceeds by two consecutive transesterification reactions via a lariat–intron intermediate. Here we present the 3.8 Å cryo-electron microscopy structure of the spliceosome immediately after lariat formation. The 5′-splice site is cleaved but remains close to the catalytic Mg2+ site in the U2/U6 small nuclear RNA (snRNA) triplex, and the 5′-phosphate of the intron nucleotide G(+1) is linked to the branch adenosine 2′OH. The 5′-exon is held between the Prp8 amino-terminal and linker domains, and base-pairs with U5 snRNA loop 1. Non-Watson–Crick interactions between the branch helix and 5′-splice site dock the branch adenosine into the active site, while intron nucleotides +3 to +6 base-pair with the U6 snRNA ACAGAGA sequence. Isy1 and the step-one factors Yju2 and Cwc25 stabilize docking of the branch helix. The intron downstream of the branch site emerges between the Prp8 reverse transcriptase and linker domains and extends towards the Prp16 helicase, suggesting a plausible mechanism of remodelling before exon ligation.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 51 print issues and online access
196,21 € per year
only 3,85 € per issue
Buy this article
- Purchase on SpringerLink
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Accession codes
Data deposits
The cryo-EM maps have been deposited in the Electron Microscopy Data Bank with accession codes EMD-4055, EMD-4056, EMD-4057, EMD-4058 and EMD-4059. The coordinates of the atomic models have been deposited in the Protein Data Bank under accession code 5LJ3 (core of the complex) and 5LJ5 (overall structure).
References
Will, C. L. & Lührmann, R. Spliceosome structure and function. Cold Spring Harb. Perspect. Biol. 3, a003707 (2011)
Burge, C. B., Tuschl, T. & Sharp, P. A. in The RNA World II (eds Gesteland, R. F., Cech, T. R. & Atkins, J. F. ) 525–560 (Cold Spring Harbor Laboratory Press, 1999)
Lambowitz, A. M. & Zimmerly, S. Group II introns: mobile ribozymes that invade DNA. Cold Spring Harb. Perspect. Biol. 3, a003616 (2011)
Chan, S.-P., Kao, D.-I., Tsai, W.-Y. & Cheng, S.-C. The Prp19p-associated complex in spliceosome activation. Science 302, 279–282 (2003)
Ohi, M. D. & Gould, K. L. Characterization of interactions among the Cef1p-Prp19p-associated splicing complex. RNA 8, 798–815 (2002)
Fabrizio, P. et al. The evolutionarily conserved core design of the catalytic activation step of the yeast spliceosome. Mol. Cell 36, 593–608 (2009)
Lesser, C. F. & Guthrie, C. Mutations in U6 snRNA that alter splice site specificity: implications for the active site. Science 262, 1982–1988 (1993)
Kandels-Lewis, S. & Séraphin, B. Involvement of U6 snRNA in 5′ splice site selection. Science 262, 2035–2039 (1993)
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)
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)
Newman, A. J. & Norman, C. U5 snRNA interacts with exon sequences at 5′ and 3′ splice sites. Cell 68, 743–754 (1992)
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)
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)
Cordin, O., Hahn, D. & Beggs, J. D. Structure, function and regulation of spliceosomal RNA helicases. Curr. Opin. Cell Biol. 24, 431–438 (2012)
Schwer, B. A conformational rearrangement in the spliceosome sets the stage for Prp22-dependent mRNA release. Mol. Cell 30, 743–754 (2008)
Company, M., Arenas, J. & Abelson, J. Requirement of the RNA helicase-like protein PRP22 for release of messenger RNA from spliceosomes. Nature 349, 487–493 (1991)
Tsai, R.-T. et al. Spliceosome disassembly catalyzed by Prp43 and its associated components Ntr1 and Ntr2. Genes Dev. 19, 2991–3003 (2005)
Abelson, J. et al. Conformational dynamics of single pre-mRNA molecules during in vitro splicing. Nat. Struct. Mol. Biol. 17, 504–512 (2010)
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)
Wan, R. et al. The 3.8 Å structure of the U4/U6.U5 tri-snRNP: Insights into spliceosome assembly and catalysis. Science 351, 466–475 (2016)
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)
Rasche, N. et al. Cwc2 and its human homologue RBM22 promote an active conformation of the spliceosome catalytic centre. EMBO J. 31, 1591–1604 (2012)
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)
Perriman, R. J. & Ares, M., Jr. Rearrangement of competing U2 RNA helices within the spliceosome promotes multiple steps in splicing. Genes Dev. 21, 811–820 (2007)
Grainger, R. J., Barrass, J. D., Jacquier, A., Rain, J.-C. & Beggs, J. D. Physical and genetic interactions of yeast Cwc21p, an ortholog of human SRm300/SRRM2, suggest a role at the catalytic center of the spliceosome. RNA 15, 2161–2173 (2009)
Yan, C. et al. Structure of a yeast spliceosome at 3.6-angstrom resolution. Science 349, 1182–1191 (2015)
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)
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)
Smith, D. J., Query, C. C. & Konarska, M. M. “Nought may endure but mutability”: spliceosome dynamics and the regulation of splicing. Mol. Cell 30, 657–666 (2008)
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)
Kim, C. H. & Abelson, J. Site-specific crosslinks of yeast U6 snRNA to the pre-mRNA near the 5′ splice site. RNA 2, 995–1010 (1996)
Buchwald, G., Schüssler, S., Basquin, C., Le Hir, H. & Conti, E. Crystal structure of the human eIF4AIII-CWC22 complex shows how a DEAD-box protein is inhibited by a MIF4G domain. Proc. Natl Acad. Sci. USA 110, E4611–E4618 (2013)
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)
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)
Steitz, T. A. & Steitz, J. A. A general two-metal-ion mechanism for catalytic RNA. Proc. Natl Acad. Sci. USA 90, 6498–6502 (1993)
Fica, S. M. et al. RNA catalyses nuclear pre-mRNA splicing. Nature 503, 229–234 (2013)
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)
Chen, H.-C., Tseng, C.-K., Tsai, R.-T., Chung, C.-S. & Cheng, S.-C. Link of NTR-mediated spliceosome disassembly with DEAH-box ATPases Prp2, Prp16, and Prp22. Mol. Cell. Biol. 33, 514–525 (2013)
Warkocki, Z. et al. Reconstitution of both steps of Saccharomyces cerevisiae splicing with purified spliceosomal components. Nat. Struct. Mol. Biol. 16, 1237–1243 (2009)
Chiu, Y.-F. et al. Cwc25 is a novel splicing factor required after Prp2 and Yju2 to facilitate the first catalytic reaction. Mol. Cell. Biol. 29, 5671–5678 (2009)
Krishnan, R. et al. Biased Brownian ratcheting leads to pre-mRNA remodeling and capture prior to first-step splicing. Nat. Struct. Mol. Biol. 20, 1450–1457 (2013)
Voorhees, R. M., Weixlbaumer, A., Loakes, D., Kelley, A. C. & Ramakrishnan, V. Insights into substrate stabilization from snapshots of the peptidyl transferase center of the intact 70S ribosome. Nat. Struct. Mol. Biol. 16, 528–533 (2009)
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)
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)
Schwer, B. & Guthrie, C. A conformational rearrangement in the spliceosome is dependent on PRP16 and ATP hydrolysis. EMBO J. 11, 5033–5039 (1992)
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)
Umen, J. G. & Guthrie, C. Prp16p, Slu7p, and Prp8p interact with the 3′ splice site in two distinct stages during the second catalytic step of pre-mRNA splicing. RNA 1, 584–597 (1995)
Chen, W. et al. Endogenous U2·U5·U6 snRNA complexes in S. pombe are intron lariat spliceosomes. RNA 20, 308–320 (2014)
Nguyen, T. H. D. et al. CryoEM structures of two spliceosomal complexes: starter and dessert at the spliceosome feast. Curr. Opin. Struct. Biol. 36, 48–57 (2016)
Garrey, S. M. et al. A homolog of lariat-debranching enzyme modulates turnover of branched RNA. RNA 20, 1337–1348 (2014)
Schreieck, A. et al. RNA polymerase II termination involves C-terminal-domain tyrosine dephosphorylation by CPF subunit Glc7. Nat. Struct. Mol. Biol. 21, 175–179 (2014)
Lin, R. J., Newman, A. J., Cheng, S. C. & Abelson, J. Yeast mRNA splicing in vitro. J. Biol. Chem. 260, 14780–14792 (1985)
Zhou, Z., Licklider, L. J., Gygi, S. P. & Reed, R. Comprehensive proteomic analysis of the human spliceosome. Nature 419, 182–185 (2002)
Hardin, J. W., Warnasooriya, C., Kondo, Y., Nagai, K. & Rueda, D. Assembly and dynamics of the U4/U6 di-snRNP by single-molecule FRET. Nucleic Acids Res. 43, 10963–10974 (2015)
Nguyen, T. H. D. et al. The architecture of the spliceosomal U4/U6.U5 tri-snRNP. Nature 523, 47–52 (2015)
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)
Rohou, A. & Grigorieff, N. CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015)
Scheres, S. H. W. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012)
Scheres, S. H. W. Semi-automated selection of cryo-EM particles in RELION-1.3. J. Struct. Biol. 189, 114–122 (2015)
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, 1485 (2015)
Scheres, S. H. W. & Chen, S. Prevention of overfitting in cryo-EM structure determination. Nat. Methods 9, 853–854 (2012)
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)
Kucukelbir, A., Sigworth, F. J. & Tagare, H. D. Quantifying the local resolution of cryo-EM density maps. Nat. Methods 11, 63–65 (2014)
Lu, P. et al. Structure of the mRNA splicing complex component Cwc2: insights into RNA recognition. Biochem. J. 441, 591–597 (2012)
van Roon, A.-M. M. et al. 113Cd NMR experiments reveal an unusual metal cluster in the solution structure of the yeast splicing protein Bud31p. Angew. Chem. Int. Edn Engl. 54, 4861–4864 (2015)
Biasini, M. et al. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res. 42, W252–W258 (2014)
Kurowski, M. A. & Bujnicki, J. M. GeneSilico protein structure prediction meta-server. Nucleic Acids Res. 31, 3305–3307 (2003)
Price, S. R., Evans, P. R. & Nagai, K. Crystal structure of the spliceosomal U2B"-U2A′ protein complex bound to a fragment of U2 small nuclear RNA. Nature 394, 645–650 (1998)
Kudla, G., Granneman, S., Hahn, D., Beggs, J. D. & Tollervey, D. Cross-linking, ligation, and sequencing of hybrids reveals RNA-RNA interactions in yeast. Proc. Natl Acad. Sci. USA 108, 10010–10015 (2011)
Yang, J. et al. The I-TASSER Suite: protein structure and function prediction. Nat. Methods 12, 7–8 (2015)
Walbott, H. et al. Prp43p contains a processive helicase structural architecture with a specific regulatory domain. EMBO J. 29, 2194–2204 (2010)
Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997)
Nicholls, R. A., Fischer, M., McNicholas, S. & Murshudov, G. N. Conformation-independent structural comparison of macromolecules with ProSMART. Acta Crystallogr. D Biol. Crystallogr. 70, 2487–2499 (2014)
Brown, A. et al. Tools for macromolecular model building and refinement into electron cryo-microscopy reconstructions. Acta Crystallogr. D Biol. Crystallogr. 71, 136–153 (2015)
Goddard, T. D., Huang, C. C. & Ferrin, T. E. Visualizing density maps with UCSF Chimera. J. Struct. Biol. 157, 281–287 (2007)
Marcia, M. & Pyle, A. M. Visualizing group II intron catalysis through the stages of splicing. Cell 151, 497–507 (2012)
Le Hir, H., Saulière, J. & Wang, Z. The exon junction complex as a node of post-transcriptional networks. Nat. Rev. Mol. Cell Biol. 17, 41–54 (2016)
Bessonov, S., Anokhina, M., Will, C. L., Urlaub, H. & Lührmann, R. Isolation of an active step I spliceosome and composition of its RNP core. Nature 452, 846–850 (2008)
Alexandrov, A., Colognori, D., Shu, M.-D. & Steitz, J. A. Human spliceosomal protein CWC22 plays a role in coupling splicing to exon junction complex deposition and nonsense-mediated decay. Proc. Natl Acad. Sci. USA 109, 21313–21318 (2012)
Steckelberg, A.-L., Boehm, V., Gromadzka, A. M. & Gehring, N. H. CWC22 connects pre-mRNA splicing and exon junction complex assembly. Cell Reports 2, 454–461 (2012)
Barbosa, I. et al. Human CWC22 escorts the helicase eIF4AIII to spliceosomes and promotes exon junction complex assembly. Nat. Struct. Mol. Biol. 19, 983–990 (2012)
Bono, F., Ebert, J., Lorentzen, E. & Conti, E. The crystal structure of the exon junction complex reveals how it maintains a stable grip on mRNA. Cell 126, 713–725 (2006)
Andersen, C. B. F. et al. Structure of the exon junction core complex with a trapped DEAD-box ATPase bound to RNA. Science 313, 1968–1972 (2006)
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)
Acknowledgements
We thank K. Nguyen, X.-C. Bai and S. Scheres for their 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 mass spectrometry facility for help with protein identification, A. Brown, P. Emsley, G. Murshudov and J. Llacer for help and advice with model building and refinement; L. Langer and 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, S. Scheres, C. Plaschka 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). M.E.W. was supported by a Rutherford Memorial Cambridge Scholarship, S.M.F. by EMBO and Marie Skłodowska-Curie fellowships.
Author information
Authors and Affiliations
Contributions
W.P.G., M.E.W. and S.M.F. established experimental procedures; W.P.G. and M.E.W. prepared the sample and grids, and processed EM data. W.P.G., M.E.W. and S.M.F. collected EM data. W.P.G., M.E.W. and C.O. carried out model building and refinement. W.P.G., M.E.W., S.M.F., C.O. and K.N. analysed the structure. A.J.N. contributed to the project through his knowledge and experience on yeast splicing. Manuscript was written by W.P.G., M.E.W. and K.N. and finalized with input from all authors. K.N. initiated and orchestrated the spliceosome project.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Extended data figures and tables
Extended Data Figure 1 Biochemical characterization of the complex and initial cryo-EM analysis.
a, SDS–PAGE analysis of the purified sample. Protein identities were confirmed by mass spectrometry analysis. Protein labels are coloured according to sub-complex identity (dark blue, U5 snRNP; light blue, helicase module; orange, NTC; yellow, NTR; green, U2 snRNP; purple, splicing factors; grey, not found in density). b, Analysis of the fluorescently labelled substrate in the sample by denaturing PAGE, showing conversion of linear pre-mRNA (time point 0’) into branched lariat-intron intermediate (time point 30’), which is a predominant species in the purified sample (C complex). The two hairpins on the right depict the 2 × MS2 stem–loops attached to the 5′ end of the UBC4 pre-mRNA substrate for affinity purification. c, A typical cryo-EM micrograph collected on an FEI Titan Krios microscope operated at 300 kV and detected with a Gatan K2 Summit camera. d, Reference-free 2D classification results. e, Detail of a single class average with major domains labelled.
Extended Data Figure 2 Overview of the data processing scheme used in this study.
Iterative 2D classification, template selection and automated particle picking resulted in 248K particles which were classified in 3D with a scaled and low-pass-filtered model of ILS (EMDB-6413) as a reference. The best class was refined to 3.8 Å resolution overall. Focused classification allowed us to obtain two other maps with improved quality of the peripheral regions (Prp19 and helicase modules, EMD-4056 and EMD-4057). Classification of the core complex with fine angular sampling and local searches revealed a subtle movement of the U2 snRNP which correlates with the appearance of the extra density, interpreted as a WD40 domain which belongs to Prp17 or Prp19.
Extended Data Figure 3 Global and local resolution analysis.
a, Two orthogonal sections through the map showing variation in the local resolution as estimated by Resmap. b, An overall map of the core complex c, Gold-standard FSC plots for three maps used in this study. d, Map of the core complex with a helicase module. e, A map of the core complex with Prp19 module.
Extended Data Figure 4 Examples of cryo-EM density at the core of the complex with atomic models built in.
a, U5 snRNA loop 1 with 5′-exon bound. b, The active site with exon, intron, U2 and U6 snRNAs. c, Two helices of the Prp8 reverse transcriptase thumb/X domain, showing a clear helical pitch and excellent densities for the side chains. d, 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 two half maps. Excellent correlation up to 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 Metal binding by the catalytic core of C complex.
a, b, Structure (a) and schematic representation (b) of the active site of a group IIC intron trapped in the pre-catalytic state in the presence of Ca2+ (PDB 4FAQ, ref. 76). The 5′ splice site scissile phosphate is aligned with the two metals bound at the core in a catalytic configuration, as shown in b. Note that, in this pre-catalytic structure, the group II domain VI is not present and therefore the structure does not contain the bulged adenosine nucleophile required for the branching reaction. As a result, the nucleophile is a water molecule, rather than the 2′-OH of the branch site adenosine found in spliceosomal introns. c, d, e, Structure of the RNA at the active site of spliceosomal C complex, showing the overall architecture (c), schematic of metal binding (d), and comparison of the model with the EM density (e). Note conservation of the metal binding residues compared to the group II intron (compare with ref. 36) and proximity of the cleaved G(−1)–G(+1) bond to putative M1. f, Proposed interactions between U6 snRNA and the two catalytic Mg2+ during the transition state for branching, as inferred from biochemistry36. g, h, Structure (g) and schematic (h) of the RNA core of the U2.U6.U5 ILS complex in a post-catalytic configuration (PDB 3JB9, ref. 26), probably following release of the mRNA. The two Mg2+ are shown as modelled in the coordinates deposited by the authors of the ILS structure (PDB 3JB9, ref. 26). In the ILS structure M1 and M2 are further apart (7.2 Å) than in most other structures of RNAs that coordinate catalytic metals (usually 3.9–5 Å); nonetheless, the ligands modelled for M1 and M2 are consistent with the ligands identified biochemically for the two catalytic Mg2+ necessary for splicing (compare PDB 3JB9 and 4R0D with the data in refs 34,36). Note that the branch helix is undocked from the U6 snRNA metal binding site and G(+1) is far away from the two Mg2+ at the core. The substrate and snRNAs are colour-coded while residues that position the catalytic metals are shown in magenta.
Extended Data Figure 6 Examples of the structures of isolated components.
De novo-built proteins are shown in cartoon form, along with a secondary structure diagram for the novel zinc-finger fold of Yju2. Proteins that were modelled into low-resolution regions by rigid-body docking of crystal structures or homology models (Prp19 module, Brr2, Prp16, Prp8Jab1/MPN) are shown in their cryo-EM densities.
Extended Data Figure 7 Conformational changes between U4/U6.U5 tri-snRNP, complex C and intron–lariat spliceosome.
a, Rearrangement of the RNaseH-like domain with respect to the main body of Prp8 in all three complexes. b, α-Finger (1,575–1,598) contacting the key RNA and proteins in a context-dependent manner. c, Prp8 N-terminal domain movements along with Prp8 residues 1,406–1,436 transiently docking on top of the 5′-exon and Cwc21 in complex C, stabilizing the 5′-exon and interdomain contacts in Prp8. d, Conformational rearrangements between complex C and S. pombe ILS26 showing a coupled movement of the U2 snRNP, Syf1 and Prp19.
Extended Data Figure 8 Implications for deposition of the exon–junction complex.
In higher eukaryotes exon–junction complexes (EJCs) are deposited 20–24 nucleotides (nt) upstream of splice junctions, and form a binding platform for factors involved in nuclear export, translation, alternative splicing and nonsense-mediated mRNA decay77. The core EJC components eIF4AIII, MAGOH and Y14 are found in human B and C complexes78. Cwc22 is required for eIF4AIII recruitment to spliceosomes79,80,81 and holds it in an open, inactive conformation32. a, Crystal structure of the eIF4AIII–Cwc22 complex32 docked onto the spliceosomal C complex via superposition on Cwc22. b, Crystal structure of the core EJC82,83 superimposed on the previous model via the second RecA domain of eIF4AIII. c, The 5′-exon exiting the channel at the interface between the Prp8 Large and N-terminal domains is positioned perfectly for the deposition of the EJC, explaining how the Cwc22 MIF4G domain is involved in determining the distance of EJC deposition from the splice junction.
Supplementary information
Supplementary Data
This zipped file contains the pymol session file of the PDB coordinate. (ZIP 4213 kb)
Rights and permissions
About this article
Cite this article
Galej, W., Wilkinson, M., Fica, S. et al. Cryo-EM structure of the spliceosome immediately after branching. Nature 537, 197–201 (2016). https://doi.org/10.1038/nature19316
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nature19316
This article is cited by
-
Monovalent metal ion binding promotes the first transesterification reaction in the spliceosome
Nature Communications (2023)
-
Structural basis of catalytic activation in human splicing
Nature (2023)
-
RNA recognition by Npl3p reveals U2 snRNA-binding compatible with a chaperone role during splicing
Nature Communications (2023)
-
psiCLIP reveals dynamic RNA binding by DEAH-box helicases before and after exon ligation
Nature Communications (2021)
-
The integral spliceosomal component CWC15 is required for development in Arabidopsis
Scientific Reports (2020)