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.

  • Article
  • Published:

Structural insights into branch site proofreading by human spliceosome

Nature Structural & Molecular Biology (2024)Cite this article

Subjects

Abstract

Selection of the pre-mRNA branch site (BS) by the U2 small nuclear ribonucleoprotein (snRNP) is crucial to prespliceosome (A complex) assembly. The RNA helicase PRP5 proofreads BS selection but the underlying mechanism remains unclear. Here we report the atomic structures of two sequential complexes leading to prespliceosome assembly: human 17S U2 snRNP and a cross-exon pre-A complex. PRP5 is anchored on 17S U2 snRNP mainly through occupation of the RNA path of SF3B1 by an acidic loop of PRP5; the helicase domain of PRP5 associates with U2 snRNA; the BS-interacting stem-loop (BSL) of U2 snRNA is shielded by TAT-SF1, unable to engage the BS. In the pre-A complex, an initial U2–BS duplex is formed; the translocated helicase domain of PRP5 stays with U2 snRNA and the acidic loop still occupies the RNA path. The pre-A conformation is specifically stabilized by the splicing factors SF1, DNAJC8 and SF3A2. Cancer-derived mutations in SF3B1 damage its association with PRP5, compromising BS proofreading. Together, these findings reveal key insights into prespliceosome assembly and BS selection or proofreading by PRP5.

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

Fig. 1: Cryo-EM structures of 17S human U2 snRNP and the human pre-A spliceosome.
Fig. 2: PRP5 is anchored to 17S U2 snRNP through its N-terminal acidic loop.
Fig. 3: Recognition of the BSL of U2 snRNA.
Fig. 4: Formation and stabilization of an initial U2–BS duplex in the human pre-A complex.
Fig. 5: DNAJC8 in the pre-A complex.
Fig. 6: Cancer-derived mutations in the RNA path of SF3B1 impair its interaction with PRP5.
Fig. 7: A working model for assembly of the A complex and PRP5-mediated proofreading of BS.

Similar content being viewed by others

Data availability

The atomic coordinates for human 17S U2 snRNP and the pre-A complex have been deposited in the Protein Data Bank (PDB) under accession codes PDB 7EVO and PDB 7VPX, respectively. The EM maps of 17S U2 snRNP core region, PRP5-α1, PRP5-α2, TAT-SF1 and BSL have been deposited in the EMDB with accession codes EMD-31334, EMD-31335, EMD-31336, EMD-31337 and EMD-31338, respectively. The EM map of the pre-A complex core region, DNAJC8 region and U1 snRNP region have been deposited in EMDB with accession codes EMD-32074, EMD-32075 and EMD-32076, respectively. The accession number for the RNA sequencing data reported in this paper is from the Gene Expression Omnibus (GEO GSE185713). Source data are provided with this paper.

References

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

    Article  CAS  PubMed  Google Scholar 

  2. Yan, C., Wan, R. & Shi, Y. Molecular mechanisms of pre-mRNA splicing through structural biology of the spliceosome. Cold Spring Harb. Perspect. Biol. 11, a032409 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Das, R., Zhou, Z. L. & Reed, R. Functional association of U2 snRNP with the ATP-independent spliceosomal complex E. Mol. Cell 5, 779–787 (2000).

    Article  CAS  PubMed  Google Scholar 

  4. Ruby, S. W., Chang, T. H. & Abelson, J. Four yeast spliceosomal proteins (PRP5, PRP9, PRP11, and PRP21) interact to promote U2 snRNP binding to pre-mRNA. Genes Dev. 7, 1909–1925 (1993).

    Article  CAS  PubMed  Google Scholar 

  5. Xu, Y. Z. et al. Prp5 bridges U1 and U2 snRNPs and enables stable U2 snRNP association with intron RNA. EMBO J. 23, 376–385 (2004).

    Article  PubMed  PubMed Central  Google Scholar 

  6. Liang, W. W. & Cheng, S. C. A novel mechanism for Prp5 function in prespliceosome formation and proofreading the branch site sequence. Gene Dev. 29, 81–93 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  7. Wang, E. T. et al. Alternative isoform regulation in human tissue transcriptomes. Nature 456, 470–476 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Chen, M. & Manley, J. L. Mechanisms of alternative splicing regulation: insights from molecular and genomics approaches. Nat. Rev. Mol. Cell Biol. 10, 741–754 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Lee, Y. & Rio, D. C. Mechanisms and regulation of alternative pre-mRNA splicing. Annu. Rev. Biochem. 84, 291–323 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Bonnal, S. C., Lopez-Oreja, I. & Valcarcel, J. Roles and mechanisms of alternative splicing in cancer—implications for care. Nat. Rev. Clin. Oncol. 17, 457–474 (2020).

    Article  PubMed  Google Scholar 

  11. Smith, C. W. J. & Valcarcel, J. Alternative pre-mRNA splicing: the logic of combinatorial control. Trends Biochem. Sci. 25, 381–388 (2000).

    Article  CAS  PubMed  Google Scholar 

  12. O’Day, C. L., Dalbadie-McFarland, G. & Abelson, J. The Saccharomyces cerevisiae Prp5 protein has RNA-dependent ATPase activity with specificity for U2 small nuclear RNA. J. Biol. Chem. 271, 33261–33267 (1996).

    Article  PubMed  Google Scholar 

  13. Abu Dayyeh, B. K., Quan, T. K., Castro, M. & Ruby, S. W. Probing interactions between the U2 small nuclear ribonucleoprotein and the DEAD-box protein, Prp5. J. Biol. Chem. 277, 20221–20233 (2002).

    Article  PubMed  Google Scholar 

  14. Perriman, R., Barta, I., Voeltz, G. K., Abelson, J. & Ares, M. Jr. ATP requirement for Prp5p function is determined by Cus2p and the structure of U2 small nuclear RNA. Proc. Natl Acad. Sci. USA 100, 13857–13862 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Yang, F. et al. Mechanisms of the RNA helicases DDX42 and DDX46 in human U2 snRNP assembly. Nat. Commun. 14, 897 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Zhang, Z. W. Molecular architecture of the human 17S U2 snRNP. Nature 583, 310–313 (2020).

    Article  CAS  PubMed  Google Scholar 

  17. Perriman, R. & Ares, M. Invariant U2 snRNA nucleotides form a stem loop to recognize the intron early in splicing. Mol. Cell 38, 416–427 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Tang, Q. et al. SF3B1/Hsh155 HEAT motif mutations affect interaction with the spliceosomal ATPase Prp5, resulting in altered branch site selectivity in pre-mRNA splicing. Gene Dev. 30, 2710–2723 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Carrocci, T. J., Zoerner, D. M., Paulson, J. C. & Hoskins, A. A. SF3b1 mutations associated with myelodysplastic syndromes alter the fidelity of branchsite selection in yeast. Nucleic Acids Res. 45, 4837–4852 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Yoshida, K. & Ogawa, S. Splicing factor mutations and cancer. WIREs RNA 5, 445–459 (2014).

    Article  CAS  PubMed  Google Scholar 

  22. Bonnal, S., Vigevani, L. & Valcarcel, J. The spliceosome as a target of novel antitumour drugs. Nat. Rev. Drug Discov. 11, 847–859 (2012).

    Article  CAS  PubMed  Google Scholar 

  23. Golas, M. M., Sander, B., Will, C. L., Luhrmann, R. & Stark, H. Molecular architecture of the multiprotein splicing factor SF3b. Science 300, 980–984 (2003).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  26. Will, C. L. et al. Characterization of novel SF3b and 17S U2 snRNP proteins, including a human Prp5p homologue and an SF3b DEAD-box protein. EMBO J. 21, 4978–4988 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Kastner, B. et al. GraFix: sample preparation for single-particle electron cryomicroscopy. Nat. Methods 5, 53–55 (2008).

    Article  CAS  PubMed  Google Scholar 

  28. Zhang, X. et al. An atomic structure of the human spliceosome. Cell 169, 918–929 (2017).

    Article  CAS  PubMed  Google Scholar 

  29. Kaida, D. et al. Spliceostatin A targets SF3b and inhibits both splicing and nuclear retention of pre-mRNA. Nat. Chem. Biol. 3, 576–583 (2007).

    Article  CAS  PubMed  Google Scholar 

  30. Berget, S. M. Exon recognition in vertebrate splicing. J. Biol. Chem. 270, 2411–2414 (1995).

    Article  CAS  PubMed  Google Scholar 

  31. Sharma, S., Kohlstaedt, L. A., Damianov, A., Rio, D. C. & Black, D. L. Polypyrimidine tract binding protein controls the transition from exon definition to an intron defined spliceosome. Nat. Struct. Mol. Biol. 15, 183–191 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Cretu, C. et al. Structural basis of intron selection by U2 snRNP in the presence of covalent inhibitors. Nat. Commun. 12, 4491 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Schneider, M. et al. Exon definition complexes contain the tri-snRNP and can be directly converted into B-like precatalytic splicing complexes. Mol. Cell 38, 223–235 (2010).

    Article  CAS  PubMed  Google Scholar 

  34. Tholen, J., Razew, M., Weis, F. & Galej, W. P. Structural basis of branch site recognition by the human spliceosome. Science 375, 50–57 (2022).

    Article  CAS  PubMed  Google Scholar 

  35. Shao, W., Kim, H. S., Cao, Y., Xu, Y. Z. & Query, C. C. A U1–U2 snRNP interaction network during intron definition. Mol. Cell. Biol. 32, 470–478 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Zhang, X. et al. Structure of the human activated spliceosome in three conformational states. Cell Res 28, 307–322 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Zhan, X., Yan, C., Zhang, X., Lei, J. & Shi, Y. Structures of the human pre-catalytic spliceosome and its precursor spliceosome. Cell Res 28, 1129–1140 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Rauhut, R. et al. Molecular architecture of the Saccharomyces cerevisiae activated spliceosome. Science 353, 1399–1405 (2016).

    Article  CAS  PubMed  Google Scholar 

  39. Cretu, C. et al. Structural basis of splicing modulation by antitumor macrolide compounds. Mol. Cell 70, 265 (2018).

    Article  CAS  PubMed  Google Scholar 

  40. Finci, L. I. et al. The cryo-EM structure of the SF3b spliceosome complex bound to a splicing modulator reveals a pre-mRNA substrate competitive mechanism of action. Genes Dev. 32, 309–320 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Nesic, D. & Kramer, A. Domains in human splicing factors SF3a60 and SF3a66 required for binding to SF3a120, assembly of the 17S U2 snRNP, and prespliceosome formation. Mol. Cell. Biol. 21, 6406–6417 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Selenko, P. et al. Structural basis for the molecular recognition between human splicing factors U2AF(65) and SF1/mBBP. Mol. Cell 11, 965–976 (2003).

    Article  CAS  PubMed  Google Scholar 

  44. Abovich, N. & Rosbash, M. Cross-intron bridging interactions in the yeast commitment complex are conserved in mammals. Cell 89, 403–412 (1997).

    Article  CAS  PubMed  Google Scholar 

  45. Crisci, A. et al. Mammalian splicing factor SF1 interacts with SURP domains of U2 snRNP-associated proteins. Nucleic Acids Res. 43, 10456–10473 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Liu, Z. H. et al. Structural basis for recognition of the intron branch site RNA by splicing factor 1. Science 294, 1098–1102 (2001).

    Article  CAS  PubMed  Google Scholar 

  47. Jacewicz, A., Chico, L., Smith, P., Schwer, B. & Shuman, S. Structural basis for recognition of intron branchpoint RNA by yeast Msl5 and selective effects of interfacial mutations on splicing of yeast pre-mRNAs. RNA 21, 401–414 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Kao, C. Y., Cao, E. C., Wai, H. L. & Cheng, S. C. Evidence for complex dynamics during U2 snRNP selection of the intron branchpoint. Nucleic Acids Res. 49, 9965–9977 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Gozani, O., Feld, R. & Reed, R. Evidence that sequence-independent binding of highly conserved U2 snRNP proteins upstream of the branch site is required for assembly of spliceosomal complex A. Gene Dev. 10, 233–243 (1996).

    Article  CAS  PubMed  Google Scholar 

  50. Ma, C. T. et al. Ordered multi-site phosphorylation of the splicing factor ASF/SF2 by SRPK1. J. Mol. Biol. 376, 55–68 (2008).

    Article  CAS  PubMed  Google Scholar 

  51. Zhong, X. Y., Ding, J. H., Adams, J. A., Ghosh, G. & Fu, X. D. Regulation of SR protein phosphorylation and alternative splicing by modulating kinetic interactions of SRPK1 with molecular chaperones. Gene Dev. 23, 482–495 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Yoshida, K. et al. Frequent pathway mutations of splicing machinery in myelodysplasia. Blood 118, 212–212 (2011).

    Article  Google Scholar 

  53. Liu, Z. Q. et al. Mutations in the RNA splicing factor SF3B1 promote tumorigenesis through MYC stabilization. Cancer Discov. 10, 806–821 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. DeBoever, C. et al. Transcriptome sequencing reveals potential mechanism of cryptic 3′ splice site selection in SF3B1-mutated cancers. PLoS Comput. Biol. 11, e1004105 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  55. Darman, R. B. et al. Cancer-associated SF3B1 hotspot mutations induce cryptic 3′ splice site selection through use of a different branch point. Cell Rep. 13, 1033–1045 (2015).

    Article  CAS  PubMed  Google Scholar 

  56. Alsafadi, S. et al. Cancer-associated SF3B1 mutations affect alternative splicing by promoting alternative branchpoint usage. Nat. Commun. 7, 10615 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Yin, S. Y. et al. A murine model of chronic lymphocytic leukemia based on B cell-restricted expression of Sf3b1 mutation and Atm deletion. Cancer Cell 35, 283 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Brooks, A. N. et al. Conservation of an RNA regulatory map between Drosophila and mammals. Genome Res. 21, 193–202 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Buonamici, S. et al. Abstract 2040: Mutations in SF3B1 lead to aberrant splicing through cryptic 3′ splice site selection and impair hematopoietic cell differentiation. Cancer Res 75, https://doi.org/10.1158/1538-7445.Am2015-2040 (2015).

  60. Zhang, Z. et al. Structural insights into how Prp5 proofreads the pre-mRNA branch site. Nature 596, 296–300 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Zeng, Y. et al. Profiling lariat intermediates reveals genetic determinants of early and late co-transcriptional splicing. Mol. Cell 82, 4681–4699 e4688 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Buonamici, S. et al. H3B-8800, an orally bioavailable modulator of the SF3b complex, shows efficacy in spliceosome-mutant myeloid malignancies. Blood 128, 966 (2016).

    Article  Google Scholar 

  63. Seiler, M. et al. H3B-8800, an orally available small-molecule splicing modulator, induces lethality in spliceosome-mutant cancers. Nat. Med. 24, 497–504 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Steensma, D. P. et al. Results of a clinical trial of H3B-8800, a splicing modulator, in patients with myelodysplastic syndromes (MDS), acute myeloid leukemia (AML) or chronic myelomonocytic leukemia (CMML). Blood 134, 673 (2019).

    Article  Google Scholar 

  65. Ghosh, A. K., Mishevich, J. L. & Jurica, M. S. Spliceostatins and derivatives: chemical syntheses and biological properties of potent splicing inhibitors. J. Nat. Prod. 84, 1681–1706 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Bai, R. et al. Mechanism of spliceosome remodeling by the ATPase/helicase Prp2 and its coactivator Spp2. Science 371, 141 (2021).

    Article  Google Scholar 

  67. Zhan, X., Yan, C., Zhang, X., Lei, J. & Shi, Y. Structure of a human catalytic step I spliceosome. Science 359, 537–545 (2018).

    Article  CAS  PubMed  Google Scholar 

  68. Chiara, M. D. et al. Identification of proteins that interact with exon sequences, splice sites, and the branchpoint sequence during each stage of spliceosome assembly. Mol. Cell. Biol. 16, 3317–3326 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Jurica, M. S., Licklider, L. J., Gygi, S. P., Grigorieff, N. & Moore, M. J. Purification and characterization of native spliceosomes suitable for three-dimensional structural analysis. RNA 8, 426–439 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Dignam, J. D., Lebovitz, R. M. & Roeder, R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11, 1475–1489 (1983).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Lei, J. L. & Frank, J. Automated acquisition of cryo-electron micrographs for single particle reconstruction on an FEI Tecnai electron microscope. J. Struct. Biol. 150, 69–80 (2005).

    Article  PubMed  Google Scholar 

  72. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Zhang, K. Gctf: real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

    Article  CAS  PubMed  Google Scholar 

  75. Chen, S. X. et al. High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy. Ultramicroscopy 135, 24–35 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Rosenthal, P. B. & Henderson, R. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721–745 (2003).

    Article  CAS  PubMed  Google Scholar 

  77. Swint-Kruse, L. & Brown, C. S. Resmap: automated representation of macromolecular interfaces as two-dimensional networks. Bioinformatics 21, 3327–3328 (2005).

    Article  CAS  PubMed  Google Scholar 

  78. Steiner, R. A. & Murshudov, G. N. Flat model bulk solvent correction in the program REFMAC. Acta Crystallogr. 56, S301 (2000).

    Article  Google Scholar 

  79. Nicholls, R. & Murshudov, G. ProSMART—procrustes structural matching alignment and restraints tool. Acta Crystallogr. 67, C745–C745 (2011).

    Article  Google Scholar 

  80. Amunts, A. et al. Structure of the yeast mitochondrial large ribosomal subunit. Science 343, 1485–1489 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).

    Article  CAS  PubMed  Google Scholar 

  82. Harrow, J. et al. GENCODE: the reference human genome annotation for The ENCODE Project. Genome Res. 22, 1760–1074 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank the Cryo-EM Facility, the Computing Center, the Crystallography Facility and the Mass Spectrometry & Metabolomics Core Facility of Westlake University for technical support. We thank N. A. Larsen from H3 Biomedicine for providing SSA. This work was supported by funds from the National Natural Science Foundation of China (31930059 to Y.S.), the China Postdoctoral Science Foundation (2020M671806 to X. Zhang; 2021M692888 to X. Zhan), the National Postdoctoral Program for Innovative Talents of China (BX20200305 to X. Zhang; BX2021268 to X. Zhan) and Start-up funds from Westlake University (to Y.S.).

Author information

Author notes

  1. Xiaofeng Zhang

    Present address: Division of Reproduction and Genetics, The First Affiliated Hospital of USTC; MOE Key Laboratory for Membraneless Organelles and Cellular Dynamics, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China

  2. These authors contributed equally: Xiaofeng Zhang, Xiechao Zhan, Tong Bian, Fenghua Yang.

Authors and Affiliations

  1. Research Center for Industries of the Future, Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University; Institute of Biology, Westlake Institute for Advanced Study, Hangzhou, Zhejiang Province, China

    Xiaofeng Zhang, Xiechao Zhan, Tong Bian, Fenghua Yang, Yichen Lu, Zhihan Xing, Rongyan Fan & Yigong Shi

  2. Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang Province, China

    Xiechao Zhan, Tong Bian, Fenghua Yang, Yichen Lu, Zhihan Xing, Rongyan Fan & Yigong Shi

  3. College of Life Sciences, Fudan University, Shanghai, China

    Tong Bian, Fenghua Yang & Yichen Lu

  4. Beijing Advanced Innovation Center for Structural Biology & Frontier Research Center for Biological Structure; Tsinghua-Peking Joint Center for Life Sciences; School of Life Sciences, Tsinghua University, Beijing, China

    Pan Li, Qiangfeng Cliff Zhang & Yigong Shi

Authors
  1. Xiaofeng Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiechao Zhan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tong Bian
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Fenghua Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Pan Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yichen Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Zhihan Xing
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Rongyan Fan
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Qiangfeng Cliff Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Yigong Shi
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Y.S. conceived the project. X. Zhang, T.B. and F.Y. designed and performed the experiments. X. Zhang prepared cryo-EM samples and collected the EM data. X. Zhan, X. Zhang, Y.L. and Z.X. processed the EM data, calculated the EM maps, and built the atomic models. F.Y., R.F., P.L. and Q.Z. performed RNA-seq data analysis. Y.S. and X. Zhang wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Xiaofeng Zhang or Yigong Shi.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Structural & Molecular Biology thanks Jeffrey Pleiss and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Sara Osman, in collaboration with the Nature Structural & Molecular Biology team.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Purification and cryo-EM reconstruction of human 17S U2 snRNP.

a, Only U2 snRNA is detectable in the purified human 17S U2 snRNP as shown in this denaturing PAGE gel. b, Analysis of the purified human 17S U2 snRNP on a silver-stained SDS-PAGE gel. The protein components were identified by mass spectrometry. c, A representative cryo-EM micrograph (upper panel) and representative 2D class averages (lower panel) of the human 17S U2 snRNP sample. The experiments were repeated independently for more than three times with similar results. d, A flow chart diagram of cryo-EM data processing for human 17S U2 snRNP. Please refer to Methods for details. e, The final reconstructions for the masked core (red), PRP5-α1 (blue), PRP5-α2 (yellow), TAT-SF1(magenta) and BSL (cyan) display average resolutions of 2.5 Å, 2.9 Å, 2.9 Å, 3.8 Å and 2.8 Å, respectively, on the basis of the FSC value of 0.143. f, Two overall views of the EM density map. The local resolutions are color-coded for different regions of U2 snRNP. g, Angular distribution of the particles used for the reconstruction. h, The Fourier-shell correlation (FSC) curves for cross-validation between the model and the cryo-EM map of human 17S U2 snRNP.

Source data

Extended Data Fig. 2 Purification and cryo-EM reconstruction of human pre-A complex.

a, Analysis of RNA components on 8% urea-PAGE gel after glycerol gradient centrifugation. The fractions that contain pre-mRNA, U1 and U2 snRNAs are indicated by a red rectangular box. Fractions 11–15, thought to contain the pre-A complex, were pooled for cryo-EM analysis. b, Analysis of the purified pre-A complex on a silver-stained SDS-PAGE gel. The protein components were identified by mass spectrometry. c, A representative cryo-EM micrograph of the pre-A complex sample. d, Representative 2D class averages. Scale bar: 44 nm. The experiments were repeated independently for more than three times with similar results. e, A flow chart diagram of cryo-EM data processing for human pre-A complex. Please refer to Methods for details. f, The final reconstruction for the masked U2 region displays an average resolution of 3.0 Å on the basis of the FSC value of 0.143. g, Two overall views of the EM density map. The local resolutions are color-coded for different regions of U2 snRNP in the pre-A complex. h, Angular distribution of the particles used for the reconstruction of U2 snRNP region of human pre-A complex. i, The Fourier-shell correlation (FSC) curves for cross-validation between the model and the cryo-EM map for the human pre-A complex.

Source data

Extended Data Fig. 3 The EM density map of human 17S U2 snRNP.

a, The EM density map for the small molecule E7107 and its surrounding structural elements. Two related views are shown. b, The EM density map for the RRM domain of the splicing factor TAT-SF1 (left panel). The EM density maps for two representative α-helices are shown (middle and right panels). c, The EM density map for the Linker domain of TAT-SF1. d, The EM density map for SF3A2. The separator helix is indicated by a black dashed box. e, The EM density map for the α1 helix of PRP5 and its interacting elements from SF3B1. Two related views are shown. f, The EM density map for the acidic loop of PRP5 and its interacting elements from SF3B1. Two related views are shown. g, The EM density map for the α2 helix of PRP5 and its interacting elements from SF3B1. Two related views are shown.

Extended Data Fig. 4 The EM density map for different regions of the human pre-A complex.

a, The EM density map for the initial U2/BS duplex in the pre-A complex. b, The EM density map for the zinc finger (ZnF) of SF3A2. c, The EM density map for the structurally resolved region of DNAJC8. Two related views are shown. d, Docking of SF1 into the EM density map. e, Docking of U1 snRNP into the EM density map. f, Superimposition of the initial U2/BS duplex from pre-A complex and the three-way junction of U2 snRNA from human 17S U2 snRNP (grey). g, The low-pass filtered density map for the 5′-end sequences of U2 snRNA can be traced to the helicase domain of PRP5.

Extended Data Fig. 5 PRP5 knockdown in HEK293F cells induces altered splicing site selection.

a, Analysis of PRP5 mRNA level (top panel) and protein expression level (bottom panel) in control (NC) and siRNA-transfected cells (mean ± s.d., n = 3 independent replicates). b, The volcano plot of log2 fold change versus log10(Pvalue) of gene expression level in siRNA-transfected cells and WT cells. Red and blue dots indicate up-regulated and down-regulated genes, respectively (Pvalue < 0.001). c, The volcano plot of log2 fold change of ∆PSI versus log10(Pvalue) of all splicing changes. Red dots indicate significantly altered splicing events (|∆PSI| > 10 and Pvalue < 0.05). d, Distribution of novel (orange) and known (grey) aberrant splicing events in PRP5 knockdown cells. Intron retention and alternative 3’SS are the most frequently occurring events. See also Extended Data Table 4. e, Validation of representative aberrantly spliced genes in control (n = 3) and PRP5 knockdown (n = 5) cells through quantitative RT-PCR assays. f, A schematic diagram of normal and aberrant splicing of ANKHD1, a representative gene that is sensitive to PRP5 knockdown. Positions of the cryptic (red) and canonical (green) 3’SS are indicated. P values were calculated by a two-sided two-test without adjustment.

Source data

Extended Data Fig. 6 The open and closed conformations of SF3B1.

a, The open conformation of SF3B1. SF3B1 exists in an open conformation in both 17S U2 snRNP and the pre-A complex. Shown here is an overlay of the SF3B1 structures from these two complexes. b, The closed conformation of SF3B1. Pre-mRNA engagement by U2 snRNP results in a closed conformation of SF3B1. Shown here is the structure of SF3B1 from the human Bact complex. c, Structural alignment using HEAT repeats 16 through 20 of SF3B1 reveals major conformational differences between the open and closed states of SF3B1. Two perpendicular views are shown. d, Structural alignment using HEAT repeats 4 through 9 of SF3B1 reveals major conformational differences between the open and closed states of SF3B1.

Extended Data Fig. 7 Structural comparison between the yeast intron-defined pre-A complex and the human exon-defined pre-A complex.

a, Structure of the pre-A complex from S. cerevisiae. The yeast complex was assembled on an intron-definition pre-mRNA. A schematic diagram of the pre-mRNA and its recognition by the pre-A complex is shown above. b, Structure of the human pre-A complex. The human complex was assembled on an exon-definition pre-mRNA. A schematic diagram of the pre-mRNA and its recognition by the pre-A complex is shown above.

Extended Data Fig. 8 The relative orientations of U1 and U2 snRNPs in yeast and human spliceosomes.

a, Molecular organizations of yeast U1 (pink) and U2 (green and light blue) snRNPs in the pre-A, A and pre-B complexes. Overall, the U1 snRNP moves away from U2 snRNP to allow the recruitment of tri-snRNP. b, In human, the cross-exon pre-A complex is expected to be converted to cross-intron complex for splicing reaction to proceed; this step may involve interaction of U2 snRNP with another U1 snRNP that bound an upstream 5′SS.

Extended Data Fig. 9 Recognition of the splicing inhibitors E7107 and SSA by the Hinged pocket.

a, A close-up view on the EM density of E7107 in human 17S U2 snRNP. b, A close-up view on the interface between E7107 and the Hinged pocket. The Hinged pocket is exactly where the BPA binds in the assembled spliceosomes. c, A close-up view on the EM density of SSA in the human pre-A complex. SSA is covalently linked to Cys26 of PHF5A. d, A close-up view on the interface between SSA and the Hinged pocket.

Supplementary information

Supplementary Information

Supplementary Tables 1–3.

Reporting Summary

Supplementary Table 4

List of aberrant splicing events associated with PRP5 knockdown in HEK293F cells.

Source data

Source Data Fig. 6

Unprocessed western blots.

Source Data Extended Data Fig. 1

Unprocessed gels.

Source Data Extended Data Fig. 2

Unprocessed gels.

Source Data Extended Data Fig. 5

Unprocessed western blots.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhang, X., Zhan, X., Bian, T. et al. Structural insights into branch site proofreading by human spliceosome. Nat Struct Mol Biol (2024). https://doi.org/10.1038/s41594-023-01188-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/s41594-023-01188-0

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