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.

  • Review Article
  • Published:

Cryo-electron microscopy snapshots of the spliceosome: structural insights into a dynamic ribonucleoprotein machine

Nature Structural & Molecular Biology volume 24, pages 791–799 (2017)Cite this article

Subjects

Abstract

The spliceosome excises introns from pre-messenger RNAs using an RNA-based active site that is cradled by a dynamic protein scaffold. A recent revolution in cryo-electron microscopy (cryo-EM) has led to near-atomic-resolution structures of key spliceosome complexes that provide insight into the mechanism of activation, splice site positioning, catalysis, protein rearrangements and ATPase-mediated dynamics of the active site. The cryo-EM structures rationalize decades of observations from genetic and biochemical studies and provide a molecular framework for future functional studies.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: A functional view of the splicing cycle.
Figure 2: A structural view of the splicing cycle.
Figure 3: Movement of the Prp8 RNaseH-like domain and its interaction with active site elements.
Figure 4: The active site of the spliceosome and its interaction with substrate.
Figure 5: Activation of the spliceosome.
Figure 6: Binding of DEAH-box ATPases to specific spliceosomal complexes.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Padgett, R.A., Konarska, M.M., Grabowski, P.J., Hardy, S.F. & Sharp, P.A. Lariat RNAs as intermediates and products in the splicing of messenger RNA precursors. Science 225, 898–903 (1984).

    CAS  PubMed  Google Scholar 

  2. Domdey, H. et al. Lariat structures are in vivo intermediates in yeast pre-mRNA splicing. Cell 39, 611–621 (1984).

    CAS  PubMed  Google Scholar 

  3. Ruskin, B., Krainer, A.R., Maniatis, T. & Green, M.R. Excision of an intact intron as a novel lariat structure during pre-mRNA splicing in vitro. Cell 38, 317–331 (1984).

    CAS  PubMed  Google Scholar 

  4. Rodriguez, J.R., Pikielny, C.W. & Rosbash, M. In vivo characterization of yeast mRNA processing intermediates. Cell 39, 603–610 (1984).

    CAS  PubMed  Google Scholar 

  5. Staley, J.P. & Guthrie, C. Mechanical devices of the spliceosome: motors, clocks, springs and things. Cell 92, 315–326 (1998).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  7. Séraphin, B., Kretzner, L. & Rosbash, M. A U1 snRNA:pre-mRNA base-pairing interaction is required early in yeast spliceosome assembly but does not uniquely define the 5′ cleavage site. EMBO J. 7, 2533–2538 (1988).

    PubMed  PubMed Central  Google Scholar 

  8. Zhuang, Y. & Weiner, A.M. A compensatory base change in U1 snRNA suppresses a 5′ splice site mutation. Cell 46, 827–835 (1986).

    CAS  PubMed  Google Scholar 

  9. Siliciano, P.G. & Guthrie, C. 5′ splice site selection in yeast: genetic alterations in base pairing with U1 reveal additional requirements. Genes Dev. 2, 1258–1267 (1988).

    CAS  PubMed  Google Scholar 

  10. Parker, R., Siliciano, P.G. & Guthrie, C. Recognition of the TACTAAC box during mRNA splicing in yeast involves base pairing to the U2-like snRNA. Cell 49, 229–239 (1987).

    CAS  PubMed  Google Scholar 

  11. Konarska, M.M. & Sharp, P.A. Interactions between small nuclear ribonucleoprotein particles in formation of spliceosomes. Cell 49, 763–774 (1987).

    CAS  PubMed  Google Scholar 

  12. Brow, D.A. & Guthrie, C. Spliceosomal RNA U6 is remarkably conserved from yeast to mammals. Nature 334, 213–218 (1988).

    CAS  PubMed  Google Scholar 

  13. 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).

    CAS  PubMed  Google Scholar 

  14. Boesler, C. et al. A spliceosome intermediate with loosely associated tri-snRNP accumulates in the absence of Prp28 ATPase activity. Nat. Commun. 7, 11997 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 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).

    CAS  PubMed  Google Scholar 

  17. 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).

    CAS  PubMed  Google Scholar 

  18. Lesser, C.F. & Guthrie, C. Mutations in U6 snRNA that alter splice site specificity: implications for the active site. Science 262, 1982–1988 (1993).

    CAS  PubMed  Google Scholar 

  19. Kandels-Lewis, S. & Séraphin, B. Involvement of U6 snRNA in 5′ splice site selection. Science 262, 2035–2039 (1993).

    CAS  PubMed  Google Scholar 

  20. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Chan, S.-P., Kao, D.-I., Tsai, W.-Y. & Cheng, S.-C. The Prp19p-associated complex in spliceosome activation. Science 302, 279–282 (2003).

    CAS  PubMed  Google Scholar 

  23. 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).

    CAS  PubMed  Google Scholar 

  24. Ohi, M.D. & Gould, K.L. Characterization of interactions among the Cef1p–Prp19p-associated splicing complex. RNA 8, 798–815 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 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). This biochemical study proposed that DEAH-box ATPases bind away from their spliceosomal target and pull on the intron at a distance to remodel the spliceosome.

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Warkocki, Z. et al. Reconstitution of both steps of Saccharomyces cerevisiae splicing with purified spliceosomal components. Nat. Struct. Mol. Biol. 16, 1237–1243 (2009).

    CAS  PubMed  Google Scholar 

  30. Schwer, B. & Guthrie, C. A conformational rearrangement in the spliceosome is dependent on PRP16 and ATP hydrolysis. EMBO J. 11, 5033–5039 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  33. 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).

    CAS  PubMed  Google Scholar 

  34. 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).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Tsai, R.T. et al. Spliceosome disassembly catalyzed by Prp43 and its associated components Ntr1 and Ntr2. Genes Dev. 19, 2991–3003 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Fourmann, J.-B. et al. Dissection of the factor requirements for spliceosome disassembly and the elucidation of its dissociation products using a purified splicing system. Genes Dev. 27, 413–428 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Pena, V., Liu, S., Bujnicki, J.M., Lührmann, R. & Wahl, M.C. Structure of a multipartite protein-protein interaction domain in splicing factor Prp8 and its link to retinitis pigmentosa. Mol. Cell 25, 615–624 (2007).

    CAS  PubMed  Google Scholar 

  39. Ritchie, D.B. et al. Structural elucidation of a PRP8 core domain from the heart of the spliceosome. Nat. Struct. Mol. Biol. 15, 1199–1205 (2008).

    CAS  PubMed  Google Scholar 

  40. Pena, V., Rozov, A., Fabrizio, P., Lührmann, R. & Wahl, M.C. Structure and function of an RNaseH domain at the heart of the spliceosome. EMBO J. 27, 2929–2940 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Yang, K., Zhang, L., Xu, T., Heroux, A. & Zhao, R. Crystal structure of the β-finger domain of Prp8 reveals analogy to ribosomal proteins. Proc. Natl. Acad. Sci. USA 105, 13817–13822 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Santos, K.F. et al. Structural basis for functional cooperation between tandem helicase cassettes in Brr2-mediated remodeling of the spliceosome. Proc. Natl. Acad. Sci. USA 109, 17418–17423 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Mozaffari-Jovin, S. et al. Inhibition of RNA helicase Brr2 by the C-terminal tail of the spliceosomal protein Prp8. Science 341, 80–84 (2013).

    CAS  PubMed  Google Scholar 

  45. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Pena, V. et al. Common design principles in the spliceosomal RNA helicase Brr2 and in the Hel308 DNA helicase. Mol. Cell 35, 454–466 (2009).

    CAS  PubMed  Google Scholar 

  47. Leung, A.K.W., Nagai, K. & Li, J. Structure of the spliceosomal U4 snRNP core domain and its implication for snRNP biogenesis. Nature 473, 536–539 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Zhou, L. et al. Crystal structures of the Lsm complex bound to the 3′ end sequence of U6 small nuclear RNA. Nature 506, 116–120 (2014).

    CAS  PubMed  Google Scholar 

  49. 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).

    CAS  PubMed  Google Scholar 

  50. Lin, P.-C. & Xu, R.-M. Structure and assembly of the SF3a splicing factor complex of U2 snRNP. EMBO J. 31, 1579–1590 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Cretu, C. et al. Molecular architecture of SF3b and structural consequences of its cancer-related mutations. Mol. Cell 64, 307–319 (2016).

    CAS  PubMed  Google Scholar 

  52. Pomeranz Krummel, D.A., Oubridge, C., Leung, A.K.W., Li, J. & Nagai, K. Crystal structure of human spliceosomal U1 snRNP at 5.5-Å resolution. Nature 458, 475–480 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Kondo, Y., Oubridge, C., van Roon, A.-M.M. & Nagai, K. Crystal structure of human U1 snRNP, a small nuclear ribonucleoprotein particle, reveals the mechanism of 5′ splice site recognition. eLife 4, e04986 (2015).

    PubMed Central  Google Scholar 

  54. Weber, G., Trowitzsch, S., Kastner, B., Lührmann, R. & Wahl, M.C. Functional organization of the Sm core in the crystal structure of human U1 snRNP. EMBO J. 29, 4172–4184 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  56. Sander, B. et al. Organization of core spliceosomal components U5 snRNA loop I and U4–U6 di-snRNP within U4/U6–U5 tri-snRNP as revealed by electron cryomicroscopy. Mol. Cell 24, 267–278 (2006).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  58. Jurica, M.S., Sousa, D., Moore, M.J. & Grigorieff, N. Three-dimensional structure of C complex spliceosomes by electron microscopy. Nat. Struct. Mol. Biol. 11, 265–269 (2004).

    CAS  PubMed  Google Scholar 

  59. Golas, M.M. et al. 3D cryo-EM structure of an active step I spliceosome and localization of its catalytic core. Mol. Cell 40, 927–938 (2010).

    CAS  PubMed  Google Scholar 

  60. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Cohen-Krausz, S., Sperling, R. & Sperling, J. Exploring the architecture of the intact supraspliceosome using electron microscopy. J. Mol. Biol. 368, 319–327 (2007).

    CAS  PubMed  Google Scholar 

  62. Kühlbrandt, W. Cryo-EM enters a new era. eLife 3, e03678 (2014).

    PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  64. Plaschka, C., Lin, P.-C. & Nagai, K. Structure of a pre-catalytic spliceosome. Nature 546, 617–621 (2017). This paper described the cryo-EM structure of the yeast spliceosomal B complex and provided insight into Brr2-mediated activation of the spliceosome.

    CAS  PubMed  PubMed Central  Google Scholar 

  65. Rauhut, R. et al. Molecular architecture of the Saccharomyces cerevisiae activated spliceosome. Science 353, 1399–1405 (2016). The structure of yeast spliceosome after Brr2-mediated activation, providing insight into Prp2-induced remodeling.

    CAS  PubMed  Google Scholar 

  66. 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). The 3.5-Å structure of the yeast Bact complex, which revealed that the active site is already formed but that the BP adenosine is kept 50 Å away from the active site by the U2 snRNP SF3b complex.

    CAS  PubMed  Google Scholar 

  67. Nguyen, T.H. et al. The architecture of the spliceosomal U4/U6–U5 tri-snRNP. Nature 523, 47–52 (2015)The cryo-EM structure of the yeast U4/U6–U5 tri-snRNP that provided the first pseudo-atomic model of a spliceosomal complex, which revealed the organization of protein and RNA components.

    CAS  PubMed  PubMed Central  Google Scholar 

  68. 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). This paper describes a complete atomic model of the 1.5-MDa U4/U6–U5 tri-snRNP and provides important new insights into the spliceosomal activation process that leads to the formation of the catalytic center.

    CAS  PubMed  PubMed Central  Google Scholar 

  69. 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). This paper also describes a complete atomic model of yeast U4/U6–U5 tri-snRNP and shows how U6 snRNA is kept catalytically inactive by the U4 snRNA and Prp3.

    CAS  PubMed  Google Scholar 

  70. Wu, N.-Y., Chung, C.-S. & Cheng, S.-C. Role of Cwc24 in the first catalytic step of splicing and fidelity of 5′ splice site selection. Mol. Cell. Biol. 37, e00580–e16 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  71. Galej, W.P. et al. Cryo-EM structure of the spliceosome immediately after branching. Nature 537, 197–201 (2016). This study revealed the cryo-EM structure of a catalytic spliceosome with the products of the first catalytic reaction bound to the active site, elucidated how the BP adenosine docks into the active site for catalysis and provided structural insights into Prp16-dependent remodeling of the spliceosome for exon ligation.

    CAS  PubMed  PubMed Central  Google Scholar 

  72. 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). This paper also presented the cryo-EM structure of the catalytic spliceosome after the first step of splicing at 3.4-Å resolution, revealing the interactions between the substrates and surrounding proteins in detail.

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  74. Yean, S.L., Wuenschell, G., Termini, J. & Lin, R.-J. Metal-ion coordination by U6 small nuclear RNA contributes to catalysis in the spliceosome. Nature 408, 881–884 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  75. Fica, S.M. et al. RNA catalyzes nuclear pre-mRNA splicing. Nature 503, 229–234 (2013). This study identified the U6 snRNA phosphate oxygens that coordinate catalytic metal ions for branching and exon ligations, demonstrating that both reactions are catalyzed by a single RNA-based active site.

    CAS  PubMed  PubMed Central  Google Scholar 

  76. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  77. Fica, S.M. et al. Structure of a spliceosome remodeled for exon ligation. Nature 542, 377–380 (2017). This paper presented the cryo-EM structure of a spliceosome just before the second catalytic step and showed how a Prp16-induced structural change undocks the branch helix from the active site and creates a space for the incoming 3′ exon for exon ligation.

    CAS  PubMed  PubMed Central  Google Scholar 

  78. Yan, C., Wan, R., Bai, R., Huang, G. & Shi, Y. Structure of a yeast step II catalytically activated spliceosome. Science 355, 149–155 (2017). This paper also describes the cryo-EM structure of the yeast C* spliceosome in atomic detail and provides functional insights similar to those in ref. 77.

    CAS  PubMed  Google Scholar 

  79. 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 

  80. Query, C.C. & Konarska, M.M. Suppression of multiple substrate mutations by spliceosomal prp8 alleles suggests functional correlations with ribosomal ambiguity mutants. Mol. Cell 14, 343–354 (2004).

    CAS  PubMed  Google Scholar 

  81. 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).

    CAS  PubMed  Google Scholar 

  82. Liu, L., Query, C.C. & Konarska, M.M. Opposing classes of prp8 alleles modulate the transition between the catalytic steps of pre-mRNA splicing. Nat. Struct. Mol. Biol. 14, 519–526 (2007).

    CAS  PubMed  Google Scholar 

  83. Query, C.C. & Konarska, M.M. CEF1 (CDC5) alleles modulate transitions between catalytic conformations of the spliceosome. RNA 18, 1001–1013 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  84. Burgess, S.M. & Guthrie, C. A mechanism to enhance mRNA splicing fidelity: the RNA-dependent ATPase Prp16 governs usage of a discard pathway for aberrant lariat intermediates. Cell 73, 1377–1391 (1993).

    CAS  PubMed  Google Scholar 

  85. Mayas, R.M., Maita, H. & Staley, J.P. Exon ligation is proofread by the DExD/H-box ATPase Prp22p. Nat. Struct. Mol. Biol. 13, 482–490 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  86. Xu, Y.-Z. & Query, C.C. Competition between the ATPase Prp5 and branch region-U2 snRNA pairing modulates the fidelity of spliceosome assembly. Mol. Cell 28, 838–849 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  87. Lardelli, R.M., Thompson, J.X., Yates, J.R. III & Stevens, S.W. Release of SF3 from the intron branchpoint activates the first step of pre-mRNA splicing. RNA 16, 516–528 (2010).

    PubMed  PubMed Central  Google Scholar 

  88. Wlodaver, A.M. & Staley, J.P. The DExD/H-box ATPase Prp2p destabilizes and proofreads the catalytic RNA core of the spliceosome. RNA 20, 282–294 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  89. Koodathingal, P., Novak, T., Piccirilli, J.A. & Staley, J.P. The DEAH-box ATPases Prp16 and Prp43 cooperate to proofread 5′ splice site cleavage during pre-mRNA splicing. Mol. Cell 39, 385–395 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  90. Reyes, J.L., Gustafson, E.H., Luo, H.R., Moore, M.J. & Konarska, M.M. The C-terminal region of hPrp8 interacts with the conserved GU dinucleotide at the 5′ splice site. RNA 5, 167–179 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  91. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  92. Collins, C.A. & Guthrie, C. Allele-specific genetic interactions between Prp8 and RNA active site residues suggest a function for Prp8 at the catalytic core of the spliceosome. Genes Dev. 13, 1970–1982 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  93. Yan, C. et al. Structure of a yeast spliceosome at 3.6-Å resolution. Science 349, 1182–1191 (2015). The cryo-EM structure of the S. pombe intron-lariat spliceosome revealed the structure of the active site after mRNA release and provided a first view of the active site and the Prp19-associated complex.

    CAS  PubMed  Google Scholar 

  94. Garrey, S.M. et al. A homolog of lariat-debranching enzyme modulates turnover of branched RNA. RNA 20, 1337–1348 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  95. Bertram, K. et al. Cryo-EM structure of a human spliceosome activated for step 2 of splicing. Nature 542, 318–323 (2017). The structure of a human C* spliceosome is remarkably similar to its yeast counterpart, but notably two yeast proteins, Cwc2 and Ecm2, were fused to one polypeptide.

    CAS  PubMed  Google Scholar 

  96. Zhang, X. et al. An atomic structure of the human spliceosome. Cell 169, 918–929.e14 (2017). This paper presented a high-resolution structure of a human spliceosome and uncovered mammal-specific protein factors that stabilized the human C* conformation.

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  98. Agafonov, D.E. et al. Molecular architecture of the human U4/U6–U5 tri-snRNP. Science 351, 1416–1420 (2016). The 7-Å cryo-EM structure of the human tri-snRNP shows that SAD1 tethers the BRR2 helicase at the pre-activation position and keeps it away from the substrate U4 snRNA.

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  100. Bertram, K. et al. Cryo-EM structure of a pre-catalytic human spliceosome primed for activation. Cell 170, 701–713.e11 (2017). This study presents the structure of a human B complex, revealing how the BRR2 helicase binds the U4 snRNA substrate at the same position as in the yeast B complex.

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank A. Newman, C. Plaschka, C. Charenton, L. Strittmatter and W. Galej for discussions and critical reading of the manuscript and C. Plaschka for drawing Figure 5. This work was funded by the UK Medical Research Council (grant no. MC_U105184330) and a European Research Council Advanced Grant (grant no. 693087–SPLICE3D). S.M.F. has been supported by EMBO and Marie Skłodowska-Curie fellowships.

Author information

Authors and Affiliations

  1. MRC Laboratory of Molecular Biology, Cambridge, UK

    Sebastian M Fica & Kiyoshi Nagai

Authors
  1. Sebastian M Fica
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kiyoshi Nagai
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Sebastian M Fica or Kiyoshi Nagai.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Fica, S., Nagai, K. Cryo-electron microscopy snapshots of the spliceosome: structural insights into a dynamic ribonucleoprotein machine. Nat Struct Mol Biol 24, 791–799 (2017). https://doi.org/10.1038/nsmb.3463

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.3463

This article is cited by

Search

Advanced 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