1932

Abstract

The removal of noncoding introns from pre-messenger RNA (pre-mRNA) is an essential step in eukaryotic gene expression and is catalyzed by a dynamic multi-megadalton ribonucleoprotein complex called the spliceosome. The spliceosome assembles on pre-mRNA substrates by the stepwise addition of small nuclear ribonucleoprotein particles and numerous protein factors. Extensive remodeling is required to form the RNA-based active site and to mediate the pre-mRNA branching and ligation reactions. In the past two years, cryo-electron microscopy (cryo-EM) structures of spliceosomes captured in different assembly and catalytic states have greatly advanced our understanding of its mechanism. This was made possible by long-standing efforts in the purification of spliceosome intermediates as well as recent developments in cryo-EM imaging and computational methodology. The resulting high-resolution densities allow for de novo model building in core regions of the complexes. In peripheral and less ordered regions, the combination of cross-linking, bioinformatics, biochemical, and genetic data is essential for accurate modeling. Here, we summarize these achievements and highlight the critical steps in obtaining near-atomic resolution structures of the spliceosome.

Loading

Article metrics loading...

/content/journals/10.1146/annurev-biophys-070317-033410
2018-05-20
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/biophys/47/1/annurev-biophys-070317-033410.html?itemId=/content/journals/10.1146/annurev-biophys-070317-033410&mimeType=html&fmt=ahah

Literature Cited

  1. 1.  Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW et al. 2010. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66:213–21
    [Google Scholar]
  2. 2.  Agafonov DE, Kastner B, Dybkov O, Hofele RV, Liu W-T et al. 2016. Molecular architecture of the human U4/U6.U5 tri-snRNP. Science 351:1416–20
    [Google Scholar]
  3. 3.  Agafonov DE, van Santen M, Kastner B, Dube P, Will CL et al. 2016. ATPγS stalls splicing after B complex formation but prior to spliceosome activation. RNA 22:1329–37
    [Google Scholar]
  4. 4.  Arenas JE, Abelson JN 1997. Prp43: an RNA helicase-like factor involved in spliceosome disassembly. PNAS 94:11798–802
    [Google Scholar]
  5. 5.  Azubel M, Wolf SG, Sperling J, Sperling R 2004. Three-dimensional structure of the native spliceosome by cryo-electron microscopy. Mol. Cell 15:833–39
    [Google Scholar]
  6. 6.  Bai R, Yan C, Wan R, Lei J, Shi Y 2017. Structure of the post-catalytic spliceosome from Saccharomycescerevisiae. Cell 171:1589–98.e8
    [Google Scholar]
  7. 7.  Bai X-c, Fernandez IS, McMullan G, Scheres SHW 2013. Ribosome structures to near-atomic resolution from thirty thousand cryo-EM particles. eLife 2:e00461
    [Google Scholar]
  8. 8.  Bai X-c, Rajendra E, Yang G, Shi Y, Scheres SHW 2015. Sampling the conformational space of the catalytic subunit of human γ-secretase. eLife 4:e11182
    [Google Scholar]
  9. 9.  Berget SM, Moore C, Sharp PA 1977. Spliced segments at the 5′ terminus of adenovirus 2 late mRNA. PNAS 74:3171–75
    [Google Scholar]
  10. 10.  Bernecky C, Herzog F, Baumeister W, Plitzko JM, Cramer P 2016. Structure of transcribing mammalian RNA polymerase II. Nature 529:551–54
    [Google Scholar]
  11. 11.  Bernecky C, Plitzko JM, Cramer P 2017. Structure of a transcribing RNA polymerase II–DSIF complex reveals a multidentate DNA–RNA clamp. Nat. Struct. Mol. Biol. 24:809–15
    [Google Scholar]
  12. 12.  Bertram K, Agafonov DE, Dybkov O, Haselbach D, Leelaram MN et al. 2017. Cryo-EM structure of a pre-catalytic human spliceosome primed for activation. Cell 170:701–13.e11
    [Google Scholar]
  13. 13.  Bertram K, Agafonov DE, Liu W-T, Dybkov O, Will CL et al. 2017. Cryo-EM structure of a human spliceosome activated for step 2 of splicing. Nature 542:318–23
    [Google Scholar]
  14. 14.  Biasini M, Bienert S, Waterhouse A, Arnold K, Studer G et al. 2014. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res 42:W252–58
    [Google Scholar]
  15. 15.  Black DL, Steitz JA 1986. Pre-mRNA splicing in vitro requires intact U4/U6 small nuclear ribonucleoprotein. Cell 46:697–704
    [Google Scholar]
  16. 16.  Boehringer D, Makarov EM, Sander B, Makarova OV, Kastner B et al. 2004. Three-dimensional structure of a pre-catalytic human spliceosomal complex B. Nat. Struct. Mol. Biol. 11:463–68
    [Google Scholar]
  17. 17.  Boesler C, Rigo N, Anokhina MM, Tauchert MJ, Agafonov DE et al. 2016. A spliceosome intermediate with loosely associated tri-snRNP accumulates in the absence of Prp28 ATPase activity. Nat. Commun. 7:11997
    [Google Scholar]
  18. 18.  Boland A, Martin TG, Zhang Z, Yang J, Bai X-c et al. 2017. Cryo-EM structure of a metazoan separase-securin complex at near-atomic resolution. Nat. Struct. Mol. Biol. 24:414–18
    [Google Scholar]
  19. 19.  Brilot AF, Chen JZ, Cheng A, Pan J, Harrison SC et al. 2012. Beam-induced motion of vitrified specimen on holey carbon film. J. Struct. Biol. 177:630–37
    [Google Scholar]
  20. 20.  Brow DA, Guthrie C 1988. Spliceosomal RNA U6 is remarkably conserved from yeast to mammals. Nature 334:213–18
    [Google Scholar]
  21. 21.  Burge CB, Tuschl T, Sharp PA 1999. Splicing of precursors to mRNAs by the spliceosomes. The RNA World RF Gesteland, TR Cech, JF Atkins 525–60 Cold Spring Harbor, NY: Cold Spring Harb. Lab. Press, 2nd ed..
    [Google Scholar]
  22. 22.  Cech TR. 1986. The generality of self-splicing RNA: relationship to nuclear mRNA splicing. Cell 44:207–10
    [Google Scholar]
  23. 23.  Chan S-P, Kao D-I, Tsai W-Y, Cheng S-C 2003. The Prp19p-associated complex in spliceosome activation. Science 302:279–82
    [Google Scholar]
  24. 24.  Chen W, Shulha HP, Ashar-Patel A, Yan J, Green KM et al. 2014. Endogenous U2·U5·U6 snRNA complexes in S.pombe are intron lariat spliceosomes. RNA 20:308–20
    [Google Scholar]
  25. 25.  Chow LT, Gelinas RE, Broker TR, Roberts RJ 1977. An amazing sequence arrangement at the 5′ ends of adenovirus 2 messenger RNA. Cell 12:1–8
    [Google Scholar]
  26. 26.  Company M, Arenas J, Abelson J 1991. Requirement of the RNA helicase-like protein PRP22 for release of messenger RNA from spliceosomes. Nature 349:487–93
    [Google Scholar]
  27. 27.  Cordin O, Beggs JD 2013. RNA helicases in splicing. RNA Biol 10:83–95
    [Google Scholar]
  28. 28.  Das R, Zhou Z, Reed R 2000. Functional association of U2 snRNP with the ATP-independent spliceosomal complex E. Mol. Cell 5:779–87
    [Google Scholar]
  29. 29.  Dignam JD, Lebovitz RM, Roeder RG 1983. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 11:1475–89
    [Google Scholar]
  30. 30.  Edwalds-Gilbert G, Kim DH, Kim SH, Tseng YH, Yu Y, Lin RJ 2000. Dominant negative mutants of the yeast splicing factor Prp2 map to a putative cleft region in the helicase domain of DExD/H-box proteins. RNA 6:1106–19
    [Google Scholar]
  31. 31.  Emsley P, Cowtan K 2004. Coot: model-building tools for molecular graphics. Acta Crystallogr. Sect. D Biol. Crystallogr. 60:2126–32
    [Google Scholar]
  32. 32.  Fabrizio P, Abelson J 1992. Thiophosphates in yeast U6 snRNA specifically affect pre-mRNA splicing in vitro. Nucleic Acids Res 20:3659–64
    [Google Scholar]
  33. 33.  Fabrizio P, Dannenberg J, Dube P, Kastner B, Stark H et al. 2009. The evolutionarily conserved core design of the catalytic activation step of the yeast spliceosome. Mol. Cell 36:593–608
    [Google Scholar]
  34. 34.  Faruqi AR, Henderson R 2007. Electronic detectors for electron microscopy. Curr. Opin. Struct. Biol. 17:549–55
    [Google Scholar]
  35. 35.  Faruqi AR, McMullan G 2011. Electronic detectors for electron microscopy. Q. Rev. Biophys. 44:357–90
    [Google Scholar]
  36. 36.  Fernandez-Leiro R, Scheres SH 2016. Unravelling biological macromolecules with cryo-electron microscopy. Nature 537:339–46
    [Google Scholar]
  37. 37.  Fica SM, Oubridge C, Galej WP, Wilkinson ME, Bai X-C et al. 2017. Structure of a spliceosome remodelled for exon ligation. Nature 542:377–80
    [Google Scholar]
  38. 38.  Fica SM, Tuttle N, Novak T, Li N-S, Lu J et al. 2013. RNA catalyses nuclear pre-mRNA splicing. Nature 503:229–34
    [Google Scholar]
  39. 39.  Finn RD, Attwood TK, Babbitt PC, Bateman A, Bork P et al. 2017. InterPro in 2017—beyond protein family and domain annotations. Nucleic Acids Res 45:D190–99
    [Google Scholar]
  40. 40.  Fischer N, Konevega AL, Wintermeyer W, Rodnina MV, Stark H 2010. Ribosome dynamics and tRNA movement by time-resolved electron cryomicroscopy. Nature 466:329–33
    [Google Scholar]
  41. 41.  Fleckner J, Zhang M, Valcárcel J, Green MR 1997. U2AF65 recruits a novel human DEAD box protein required for the U2 snRNP-branchpoint interaction. Genes Dev 11:1864–72
    [Google Scholar]
  42. 42.  Fourmann JB, Schmitzova J, Christian H, Urlaub H, Ficner R et al. 2013. Dissection of the factor requirements for spliceosome disassembly and the elucidation of its dissociation products using a purified splicing system. Genes Dev 27:413–28
    [Google Scholar]
  43. 43.  Galej WP, Wilkinson ME, Fica SM, Oubridge C, Newman AJ, Nagai K 2016. Cryo-EM structure of the spliceosome immediately after branching. Nature 537:197–201
    [Google Scholar]
  44. 44.  Golas MM, Sander B, Will CL, Lührmann R, Stark H 2003. Molecular architecture of the multiprotein splicing factor SF3b. Science 300:980–84
    [Google Scholar]
  45. 45.  Golas MM, Sander B, Will CL, Lührmann R, Stark H 2005. Major conformational change in the complex SF3b upon integration into the spliceosomal U11/U12 di-snRNP as revealed by electron cryomicroscopy. Mol. Cell 17:869–83
    [Google Scholar]
  46. 46.  Henderson R. 1995. The potential and limitations of neutrons, electrons and X-rays for atomic resolution microscopy of unstained biological molecules. Q. Rev. Biophys. 28:171–93
    [Google Scholar]
  47. 47.  Hoskins AA, Rodgers ML, Friedman LJ, Gelles J, Moore MJ 2016. Single molecule analysis reveals reversible and irreversible steps during spliceosome activation. eLife 5:e14166
    [Google Scholar]
  48. 48.  Jurica MS, Licklider LJ, Gygi SR, Grigorieff N, Moore MJ 2002. Purification and characterization of native spliceosomes suitable for three-dimensional structural analysis. RNA 8:426–39
    [Google Scholar]
  49. 49.  Jurica MS, Moore MJ 2003. Pre-mRNA splicing: awash in a sea of proteins. Mol. Cell 12:5–14
    [Google Scholar]
  50. 50.  Jurica MS, Sousa D, Moore MJ, Grigorieff N 2004. Three-dimensional structure of C complex spliceosomes by electron microscopy. Nat. Struct. Mol. Biol. 11:265–69
    [Google Scholar]
  51. 51.  Kastner B, Fischer N, Golas MM, Sander B, Dube P et al. 2008. GraFix: sample preparation for single-particle electron cryomicroscopy. Nat. Methods 5:53–55
    [Google Scholar]
  52. 52.  Kastner B, Kornstadt U, Bach M, Lührmann R 1992. Structure of the small nuclear RNP particle U1: identification of the two structural protuberances with RNP-antigens A and 70K. J. Cell Biol. 116:839–49
    [Google Scholar]
  53. 53.  Kataoka N, Dreyfuss G 2008. Preparation of efficient splicing extracts from whole cells, nuclei, and cytoplasmic fractions. Methods Mol. Biol. 488:357–65
    [Google Scholar]
  54. 54.  Kim D-H, Edwalds-Gilbert G, Ren C, Lin R-J 1999. A mutation in a methionine tRNA gene suppresses the prp2-1 Ts mutation and causes a pre-mRNA splicing defect in Saccharomycescerevisiae. Genetics 153:1105–15
    [Google Scholar]
  55. 55.  Kim SH, Lin RJ 1996. Spliceosome activation by PRP2 ATPase prior to the first transesterification reaction of pre-mRNA splicing. Mol. Cell. Biol. 16:6810–19
    [Google Scholar]
  56. 56.  Kimanius D, Forsberg BO, Scheres SH, Lindahl E 2016. Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2. eLife 5:e18722
    [Google Scholar]
  57. 57.  Kistler AL, Guthrie C 2001. Deletion of MUD2, the yeast homolog of U2AF65, can bypass the requirement for sub2, an essential spliceosomal ATPase. Genes Dev 15:42–49
    [Google Scholar]
  58. 58.  Knapek E, Dubochet J 1980. Beam damage to organic material is considerably reduced in cryo-electron microscopy. J. Mol. Biol. 141:147–61
    [Google Scholar]
  59. 59.  Konarska MM, Sharp PA 1986. Electrophoretic separation of complexes involved in the splicing of precursors to mRNAs. Cell 46:845–55
    [Google Scholar]
  60. 60.  Korinek A, Beck F, Baumeister W, Nickell S, Plitzko JM 2011. Computer controlled cryo-electron microscopy—TOM2 a software package for high-throughput applications. J. Struct. Biol. 175:394–405
    [Google Scholar]
  61. 61.  Kozlowski LP, Bujnicki JM 2012. MetaDisorder: a meta-server for the prediction of intrinsic disorder in proteins. BMC Bioinform 13:111
    [Google Scholar]
  62. 62.  Kuhlbrandt W. 2014. Biochemistry. The resolution revolution. Science 343:1443–44
    [Google Scholar]
  63. 63.  Laggerbauer B, Achsel T, Lührmann R 1998. The human U5–200kD DEXH-box protein unwinds U4/U6 RNA duplices in vitro. PNAS 95:4188–92
    [Google Scholar]
  64. 64.  Legrain P, Seraphin B, Rosbash M 1988. Early commitment of yeast pre-mRNA to the spliceosome pathway. Mol. Cell. Biol. 8:3755–60
    [Google Scholar]
  65. 65.  Lerner MR, Boyle JA, Mount SM, Wolin SL, Steitz JA 1980. Are snRNPs involved in splicing?. Nature 283:220–24
    [Google Scholar]
  66. 66.  Li X, Liu S, Jiang J, Zhang L, Espinosa S et al. 2017. CryoEM structure of Saccharomycescerevisiae U1 snRNP offers insight into alternative splicing. Nat. Commun. 8:1035
    [Google Scholar]
  67. 67.  Li X, Mooney P, Zheng S, Booth CR, Braunfeld MB et al. 2013. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat. Methods 10:584–90
    [Google Scholar]
  68. 68.  Liang W-W, Cheng S-C 2015. A novel mechanism for Prp5 function in prespliceosome formation and proofreading the branch site sequence. Genes Dev 29:81–93
    [Google Scholar]
  69. 69.  Lin R-J, Newman AJ, Cheng S-C, Abelson J 1985. Yeast mRNA splicing in vitro. J. Biol. Chem. 260:14780–92
    [Google Scholar]
  70. 70.  Liu S, Li X, Zhang L, Jiang J, Hill RC et al. 2017. Structure of the yeast spliceosomal postcatalytic P complex. Science 358:1278–83
    [Google Scholar]
  71. 71.  Lorenz R, Bernhart SH, Höner zu Siederdissen C, Tafer H, Flamm C et al. 2011. ViennaRNA Package 2.0. Algorithms Mol. Biol. 6:26
    [Google Scholar]
  72. 72.  Lybarger S, Beickman K, Brown V, Dembla-Rajpal N, Morey K et al. 1999. Elevated levels of a U4/U6.U5 snRNP-associated protein, Spp381p, rescue a mutant defective in spliceosome maturation. Mol. Cell. Biol. 19:577–84
    [Google Scholar]
  73. 73.  Madhani HD, Guthrie C 1992. A novel base-pairing interaction between U2 and U6 snRNAs suggests a mechanism for the catalytic activation of the spliceosome. Cell 71:803–17
    [Google Scholar]
  74. 74.  Mastronarde DN. 2005. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152:36–51
    [Google Scholar]
  75. 75.  McDowall AW, Chang J-J, Freeman R, Lepault J, Walter CA, Dubochet J 1983. Electron microscopy of frozen hydrated sections of vitreous ice and vitrified biological samples. J. Microsc. 131:1–9
    [Google Scholar]
  76. 76.  McGreevy R, Singharoy A, Li Q, Zhang J, Xu D et al. 2014. xMDFF: molecular dynamics flexible fitting of low-resolution X-ray structures. Acta Crystallogr. D Biol. Crystallogr. 70:2344–55
    [Google Scholar]
  77. 77.  McMullan G, Faruqi AR, Henderson R 2016. Direct electron detectors. Methods Enzymol 579:1–17
    [Google Scholar]
  78. 78.  Moore MJ, Sharp PA 1992. Site-specific modification of pre-mRNA: the 2′-hydroxyl groups at the splice sites. Science 256:992–97
    [Google Scholar]
  79. 79.  Mougin A, Gottschalk A, Fabrizio P, Luhrmann R, Branlant C 2002. Direct probing of RNA structure and RNA-protein interactions in purified HeLa cell's and yeast spliceosomal U4/U6.U5 tri-snRNP particles. J. Mol. Biol. 317:631–49
    [Google Scholar]
  80. 80.  Newman AJ, Lin R-J, Cheng S-C, Abelson J 1985. Molecular consequences of specific intron mutations on yeast mRNA splicing in vivo and in vitro. Cell 42:335–44
    [Google Scholar]
  81. 81.  Newman AJ, Norman C 1992. U5 snRNA interacts with exon sequences at 5′ and 3′ splice sites. Cell 68:743–54
    [Google Scholar]
  82. 82.  Nguyen THD, Galej WP, Bai X-c, Oubridge C, Newman AJ et al. 2016. Cryo-EM structure of the yeast U4/U6.U5 tri-snRNP at 3.7 Å resolution. Nature 530:298–302
    [Google Scholar]
  83. 83.  Nguyen THD, Galej WP, Bai X-c, Savva CG, Newman AJ et al. 2015. The architecture of the spliceosomal U4/U6.U5 tri-snRNP. Nature 523:47–52
    [Google Scholar]
  84. 84.  Nogales E, Louder RK, He Y 2016. Cryo-EM in the study of challenging systems: the human transcription pre-initiation complex. Curr. Opin. Struct. Biol. 40:120–27
    [Google Scholar]
  85. 85.  Nogales E, Scheres SHW 2015. Cryo-EM: a unique tool for the visualization of macromolecular complexity. Mol. Cell 58:677–89
    [Google Scholar]
  86. 86.  Ohi MD, Gould KL 2002. Characterization of interactions among the Cef1p-Prp19p-associated splicing complex. RNA 8:798–815
    [Google Scholar]
  87. 87.  Ohi MD, Ren L, Wall JS, Gould KL, Walz T 2007. Structural characterization of the fission yeast U5.U2/U6 spliceosome complex. PNAS 104:3195–200
    [Google Scholar]
  88. 88.  Ohrt T, Odenwalder P, Dannenberg J, Prior M, Warkocki Z et al. 2013. Molecular dissection of step 2 catalysis of yeast pre-mRNA splicing investigated in a purified system. RNA 19:902–15
    [Google Scholar]
  89. 89.  Padgett RA, Hardy SF, Sharp PA 1983. Splicing of adenovirus RNA in a cell-free transcription system. PNAS 80:5230–34
    [Google Scholar]
  90. 90.  Pantelic RS, Meyer JC, Kaiser U, Baumeister W, Plitzko JM 2010. Graphene oxide: a substrate for optimizing preparations of frozen-hydrated samples. J. Struct. Biol. 170:152–56
    [Google Scholar]
  91. 91.  Parker R, Guthrie C 1985. A point mutation in the conserved hexanucleotide at a yeast 5′ splice junction uncouples recognition, cleavage, and ligation. Cell 41:107–18
    [Google Scholar]
  92. 92.  Perriman R, Ares M Jr 2010. Invariant U2 snRNA nucleotides form a stem loop to recognize the intron early in splicing. Mol. Cell 38:416–27
    [Google Scholar]
  93. 93.  Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM et al. 2004. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25:1605–12
    [Google Scholar]
  94. 94.  Plaschka C, Hantsche M, Dienemann C, Burzinski C, Plitzko J, Cramer P 2016. Transcription initiation complex structures elucidate DNA opening. Nature 533:353–58
    [Google Scholar]
  95. 95.  Plaschka C, Lin P-C, Nagai K 2017. Structure of a pre-catalytic spliceosome. Nature 546:617–21
    [Google Scholar]
  96. 96.  Plumpton M, McGarvey M, Beggs JD 1994. A dominant negative mutation in the conserved RNA helicase motif ‘SAT’ causes splicing factor PRP2 to stall in spliceosomes. EMBO J 13:879–87
    [Google Scholar]
  97. 97.  Pomeranz Krummel DA, Oubridge C, Leung AK, Li J, Nagai K 2009. Crystal structure of human spliceosomal U1 snRNP at 5.5 Å resolution. Nature 458:475–80
    [Google Scholar]
  98. 98.  Popenda M, Szachniuk M, Antczak M, Purzycka KJ, Lukasiak P et al. 2012. Automated 3D structure composition for large RNAs. Nucleic Acids Res 40:e112
    [Google Scholar]
  99. 99.  Potter CS, Chu H, Frey B, Green C, Kisseberth N et al. 1999. Leginon: a system for fully automated acquisition of 1000 electron micrographs a day. Ultramicroscopy 77:153–61
    [Google Scholar]
  100. 100.  Punjani A, Rubinstein JL, Fleet DJ, Brubaker MA 2017. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14:290–96
    [Google Scholar]
  101. 101.  Pyle AM. 2016. Group II intron self-splicing. Annu. Rev. Biophys. 45:183–205
    [Google Scholar]
  102. 102.  Raghunathan PL, Guthrie C 1998. RNA unwinding in U4/U6 snRNPs requires ATP hydrolysis and the DEIH-box splicing factor Brr2. Curr. Biol. 8:847–55
    [Google Scholar]
  103. 103.  Rauhut R, Fabrizio P, Dybkov O, Hartmuth K, Pena V et al. 2016. Molecular architecture of the Saccharomycescerevisiae activated spliceosome. Science 353:1399
    [Google Scholar]
  104. 104.  Reed R. 1989. The organization of 3′ splice-site sequences in mammalian introns. Genes Dev 3:2113–23
    [Google Scholar]
  105. 105.  Rohl CA, Strauss CE, Misura KM, Baker D 2004. Protein structure prediction using Rosetta. Methods Enzymol 383:66–93
    [Google Scholar]
  106. 106.  Ruskin B, Greene JM, Green MR 1985. Cryptic branch point activation allows accurate in vitro splicing of human beta-globin intron mutants. Cell 41:833–44
    [Google Scholar]
  107. 107.  Russo CJ, Passmore LA 2014. Controlling protein adsorption on graphene for cryo-EM using low-energy hydrogen plasmas. Nat. Methods 11:649–52
    [Google Scholar]
  108. 108.  Russo CJ, Passmore LA 2014. Electron microscopy: ultrastable gold substrates for electron cryomicroscopy. Science 346:1377–80
    [Google Scholar]
  109. 109.  Sander B, Golas MM, Makarov EM, Brahms H, Kastner B et al. 2006. 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 24:267–78
    [Google Scholar]
  110. 110.  Scheres SHW. 2012. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180:519–30
    [Google Scholar]
  111. 111.  Scheres SHW. 2016. Processing of structurally heterogeneous cryo-EM data in RELION. Methods Enzymol 579:125–57
    [Google Scholar]
  112. 112.  Scheres SHW, Nagai K 2017. CryoEM structures of spliceosomal complexes reveal the molecular mechanism of pre-mRNA splicing. Curr. Opin. Struct. Biol. 46:130–39
    [Google Scholar]
  113. 113.  Schneider S, Hotz HR, Schwer B 2002. Characterization of dominant-negative mutants of the DEAH-box splicing factors Prp22 and Prp16. J. Biol. Chem. 277:15452–58
    [Google Scholar]
  114. 114.  Schwer B. 2008. A conformational rearrangement in the spliceosome sets the stage for Prp22-dependent mRNA release. Mol. Cell 30:743–54
    [Google Scholar]
  115. 115.  Schwer B, Guthrie C 1992. A conformational rearrangement in the spliceosome is dependent on PRP16 and ATP hydrolysis. EMBO J 11:5033–39
    [Google Scholar]
  116. 116.  Schwer B, Guthrie C 1992. A dominant negative mutation in a spliceosomal ATPase affects ATP hydrolysis but not binding to the spliceosome. Mol. Cell. Biol. 12:3540–47
    [Google Scholar]
  117. 117.  Sharp PA. 1985. On the origin of RNA splicing and introns. Cell 42:397–400
    [Google Scholar]
  118. 118.  Sheth N, Roca X, Hastings ML, Roeder T, Krainer AR, Sachidanandam R 2006. Comprehensive splice-site analysis using comparative genomics. Nucleic Acids Res 34:3955–67
    [Google Scholar]
  119. 119.  Sibley CR, Blazquez L, Ule J 2016. Lessons from non-canonical splicing. Nat. Rev. Genet. 17:407–21
    [Google Scholar]
  120. 120.  Spingola M, Grate L, Haussler D, Ares M Jr 1999. Genome-wide bioinformatic and molecular analysis of introns in Saccharomycescerevisiae. RNA 5:221–34
    [Google Scholar]
  121. 121.  Staley JP, Guthrie C 1999. An RNA switch at the 5′ splice site requires ATP and the DEAD box protein Prp28p. Mol. Cell 3:55–64
    [Google Scholar]
  122. 122.  Stark H, Lührmann R 2006. Cryo-electron microscopy of spliceosomal components. Annu. Rev. Biophys. Biomol. Struct. 35:435–57
    [Google Scholar]
  123. 123.  Steitz TA, Steitz JA 1993. A general two-metal-ion mechanism for catalytic RNA. PNAS 90:6498–502
    [Google Scholar]
  124. 124.  Stevens SW, Abelson J 1999. Purification of the yeast U4/U6.U5 small nuclear ribonucleoprotein particle and identification of its proteins. PNAS 96:7226–31
    [Google Scholar]
  125. 125.  Strauss EJ, Guthrie C 1994. PRP28, a ‘DEAD-box’ protein, is required for the first step of mRNA splicing in vitro. Nucleic Acids Res 22:3187–93
    [Google Scholar]
  126. 126.  Sun JS, Manley JL 1995. A novel U2-U6 snRNA structure is necessary for mammalian mRNA splicing. Genes Dev 9:843–54
    [Google Scholar]
  127. 127.  Tsai R-T, Fu R-H, Yeh F-L, Tseng C-K, Lin Y-C et al. 2005. Spliceosome disassembly catalyzed by Prp43 and its associated components Ntr1 and Ntr2. Genes Dev 19:2991–3003
    [Google Scholar]
  128. 128.  Tseng C-K, Liu H-L, Cheng S-C 2011. DEAH-box ATPase Prp16 has dual roles in remodeling of the spliceosome in catalytic steps. RNA 17:145–54
    [Google Scholar]
  129. 129.  Ulrich AK, Seeger M, Schütze T, Bartlick N, Wahl MC 2016. Scaffolding in the spliceosome via single α helices. Structure 24:1972–83
    [Google Scholar]
  130. 130.  Vagin AA, Steiner RA, Lebedev AA, Potterton L, McNicholas S et al. 2004. REFMAC5 dictionary: organization of prior chemical knowledge and guidelines for its use. Acta Crystallogr. D Biol. Crystallogr. 60:2184–95
    [Google Scholar]
  131. 131.  Vijayraghavan U, Parker R, Tamm J, Iimura Y, Rossi J et al. 1986. Mutations in conserved intron sequences affect multiple steps in the yeast splicing pathway, particularly assembly of the spliceosome. EMBO J 5:1683–95
    [Google Scholar]
  132. 132.  Wan R, Yan C, Bai R, Huang G, Shi Y 2016. Structure of a yeast catalytic step I spliceosome at 3.4 Å resolution. Science 353:895–904
    [Google Scholar]
  133. 133.  Wan R, Yan C, Bai R, Lei J, Shi Y 2017. Structure of an intron lariat spliceosome from Saccharomycescerevisiae. Cell 171:120–32.e12
    [Google Scholar]
  134. 134.  Wan R, Yan C, Bai R, Wang L, Huang M et al. 2016. The 3.8 Å structure of the U4/U6.U5 tri-snRNP: insights into spliceosome assembly and catalysis. Science 351:466–75
    [Google Scholar]
  135. 135.  Wang J, Moore PB 2017. On the interpretation of electron microscopic maps of biological macromolecules. Protein Sci 26:122–29
    [Google Scholar]
  136. 136.  Warkocki Z, Odenwalder P, Schmitzova J, Platzmann F, Stark H et al. 2009. Reconstitution of both steps of Saccharomycescerevisiae splicing with purified spliceosomal components. Nat. Struct. Mol. Biol. 16:1237–43
    [Google Scholar]
  137. 137.  Webb B, Sali A 2016. Comparative protein structure modeling using MODELLER. Curr. Protoc. Bioinform. 54:56.1–37
    [Google Scholar]
  138. 138.  Wilkinson ME, Fica SM, Galej WP, Norman CM, Newman AJ, Nagai K 2017. Postcatalytic spliceosome structure reveals mechanism of 3′-splice site selection. Science 358:1283–88
    [Google Scholar]
  139. 139.  Will CL, Lührmann R 2011. Spliceosome structure and function. Cold Spring Harb. Perspect. Biol. 3:a003707
    [Google Scholar]
  140. 140.  Wriggers W. 2012. Conventions and workflows for using Situs. Acta Crystallogr. Sect. D Biol. Crystall. 68:344–51
    [Google Scholar]
  141. 141.  Wu J, Manley JL 1989. Mammalian pre-mRNA branch site selection by U2 snRNP involves base pairing. Genes Dev 3:1553–61
    [Google Scholar]
  142. 142.  Wu S, Romfo CM, Nilsen TW, Green MR 1999. Functional recognition of the 3′ splice site AG by the splicing factor U2AF35. Nature 402:832–35
    [Google Scholar]
  143. 143.  Xu Y-Z, Newnham CM, Kameoka S, Huang T, Konarska MM, Query CC 2004. Prp5 bridges U1 and U2 snRNPs and enables stable U2 snRNP association with intron RNA. EMBO J 23:376–85
    [Google Scholar]
  144. 144.  Yan C, Hang J, Wan R, Huang M, Wong CC, Shi Y 2015. Structure of a yeast spliceosome at 3.6-angstrom resolution. Science 349:1182–91
    [Google Scholar]
  145. 145.  Yan C, Wan R, Bai R, Huang G, Shi Y 2016. Structure of a yeast activated spliceosome at 3.5 Å resolution. Science 353:904–11
    [Google Scholar]
  146. 146.  Yan C, Wan R, Bai R, Huang G, Shi Y 2017. Structure of a yeast step II catalytically activated spliceosome. Science 355:149–55
    [Google Scholar]
  147. 147.  Yang J, Yan R, Roy A, Xu D, Poisson J, Zhang Y 2015. The I-TASSER Suite: protein structure and function prediction. Nat. Methods 12:7–8
    [Google Scholar]
  148. 148.  Yu YT, Maroney PA, Darzynkiwicz E, Nilsen TW 1995. U6 snRNA function in nuclear pre-mRNA splicing: a phosphorothioate interference analysis of the U6 phosphate backbone. RNA 1:46–54
    [Google Scholar]
  149. 149.  Zhang L, Li X, Zhao R 2013. Structural analyses of the pre-mRNA splicing machinery. Protein Sci 22:677–92
    [Google Scholar]
  150. 150.  Zhang X, Yan C, Hang J, Finci LI, Lei J, Shi Y 2017. An atomic structure of the human spliceosome. Cell 169:918–29.e14
    [Google Scholar]
  151. 151.  Zhou Z, Licklider LJ, Gygi SP, Reed R 2002. Comprehensive proteomic analysis of the human spliceosome. Nature 419:182–85
    [Google Scholar]
  152. 152.  Zillmann M, Zapp ML, Berget SM 1988. Gel electrophoretic isolation of splicing complexes containing U1 small nuclear ribonucleoprotein particles. Mol. Cell. Biol. 8:814–21
    [Google Scholar]
/content/journals/10.1146/annurev-biophys-070317-033410
Loading
/content/journals/10.1146/annurev-biophys-070317-033410
Loading

Data & Media loading...

  • Article Type: Review Article
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error