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.

Crystal structure of human spliceosomal U1 snRNP at 5.5 Å resolution

Abstract

Human spliceosomal U1 small nuclear ribonucleoprotein particles (snRNPs), which consist of U1 small nuclear RNA and ten proteins, recognize the 5′ splice site within precursor messenger RNAs and initiate the assembly of the spliceosome for intron excision. An electron density map of the functional core of U1 snRNP at 5.5 Å resolution has enabled us to build the RNA and, in conjunction with site-specific labelling of individual proteins, to place the seven Sm proteins, U1-C and U1-70K into the map. Here we present the detailed structure of a spliceosomal snRNP, revealing a hierarchical network of intricate interactions between subunits. A striking feature is the amino (N)-terminal polypeptide of U1-70K, which extends over a distance of 180 Å from its RNA binding domain, wraps around the core domain consisting of the seven Sm proteins and finally contacts U1-C, which is crucial for 5′-splice-site recognition. The structure of U1 snRNP provides insights into U1 snRNP assembly and suggests a possible mechanism of 5′-splice-site recognition.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Overall fold of U1 snRNA and the U1 snRNP core domain containing the seven Sm proteins.
Figure 2: Structure of the U1 snRNP core domain.
Figure 3: A mechanism of 5′-splice-site recognition.
Figure 4: Interaction of U1-70K with SL1 of U1 snRNA, the core domain and U1-C.
Figure 5: Model of the complete human U1 snRNP.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Coordinates of the protein α-carbon and the RNA phosphorus atoms have been submitted, together with structure factors, to the Protein Data Bank under accession number 3CW1.

References

  1. 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)

    Google Scholar 

  2. Will, C. L. & Lührmann, R. in The RNA World (eds Gesteland, R. F., Cech, T. R. & Atkins, J. F.) 369–400 (Cold Spring Harbor Laboratory Press, 2006)

    Google Scholar 

  3. Yu, Y.-T., Scharl, E. C., Smith, C. M. & Steitz, J. A. in The RNA World II (eds Gesteland, R. F., Cech, T. R. & Atkins, J. F.) 487–524 (Cold Spring Harbor Laboratory Press, 1999)

    Google Scholar 

  4. Bringmann, P. & Lührmann, R. Purification of the individual snRNPs U1, U2, U5 and U4/U6 from HeLa cells and characterization of their protein constituents. EMBO J. 5, 3509–3516 (1986)

    CAS  Article  Google Scholar 

  5. Jurica, M. S. & Moore, M. J. Pre-mRNA splicing: awash in a sea of proteins. Mol. Cell 12, 5–14 (2003)

    CAS  Article  Google Scholar 

  6. Makarov, E. M. et al. Small nuclear ribonucleoprotein remodeling during catalytic activation of the spliceosome. Science 298, 2205–2208 (2002)

    ADS  CAS  Article  Google Scholar 

  7. Guthrie, C. & Patterson, B. Spliceosomal snRNAs. Annu. Rev. Genet. 22, 387–419 (1988)

    CAS  Article  Google Scholar 

  8. Griffiths-Jones, S. et al. Rfam: annotating non-coding RNAs in complete genomes. Nucleic Acids Res. 33, D121–D124 (2005)

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  10. Ruby, S. W. & Abelson, J. An early hierarchic role of U1 small nuclear ribonucleoprotein in spliceosome assembly. Science 242, 1028–1035 (1988)

    ADS  CAS  Article  Google Scholar 

  11. Séraphin, B. & Rosbash, M. Identification of functional U1 snRNA-pre-mRNA complexes committed to spliceosome assembly and splicing. Cell 59, 349–358 (1989)

    Article  Google Scholar 

  12. Kohtz, J. D. et al. Protein–protein interactions and 5′-splice-site recognition in mammalian mRNA precursors. Nature 368, 119–124 (1994)

    ADS  CAS  Article  Google Scholar 

  13. Graveley, B. R. Sorting out the complexity of SR protein functions. RNA 6, 1197–1211 (2000)

    CAS  Article  Google Scholar 

  14. Krol, A. et al. Solution structure of human U1 snRNA. Derivation of a possible three-dimensional model. Nucleic Acids Res. 18, 3803–3811 (1990)

    CAS  Article  Google Scholar 

  15. Patton, J. R. & Pederson, T. The Mr 70,000 protein of the U1 small nuclear ribonucleoprotein particle binds to the 5′ stem-loop of U1 RNA and interacts with Sm domain proteins. Proc. Natl Acad. Sci. USA 85, 747–751 (1988)

    ADS  CAS  Article  Google Scholar 

  16. Query, C. C., Bentley, R. C. & Keene, J. D. A common RNA recognition motif identified within a defined U1 RNA binding domain of the 70K U1 snRNP protein. Cell 57, 89–101 (1989)

    CAS  Article  Google Scholar 

  17. Bach, M., Krol, A. & Lührmann, R. Structure-probing of U1 snRNPs gradually depleted of the U1-specific proteins A, C and 70k. Evidence that A interacts differentially with developmentally regulated mouse U1 snRNA variants. Nucleic Acids Res. 18, 449–457 (1990)

    CAS  Article  Google Scholar 

  18. Scherly, D., Boelens, W., Dathan, N. A., van Venrooij, W. J. & Mattaj, I. W. Major determinants of the specificity of interaction between small nuclear ribonucleoproteins U1A and U2B'' and their cognate RNAs. Nature 345, 502–506 (1990)

    ADS  CAS  Article  Google Scholar 

  19. Oubridge, C., Ito, N., Evans, P. R., Teo, C. H. & Nagai, K. Crystal structure at 1.92 Å resolution of the RNA-binding domain of the U1A spliceosomal protein complexed with an RNA hairpin. Nature 372, 432–438 (1994)

    ADS  CAS  Article  Google Scholar 

  20. Nelissen, R. L., Will, C. L., van Venrooij, W. J. & Lührmann, R. The association of the U1-specific 70K and C proteins with U1 snRNPs is mediated in part by common U snRNP proteins. EMBO J. 13, 4113–4125 (1994)

    CAS  Article  Google Scholar 

  21. Muto, Y. et al. The structure and biochemical properties of the human spliceosomal protein U1C. J. Mol. Biol. 341, 185–198 (2004)

    CAS  Article  Google Scholar 

  22. Kambach, C. et al. Crystal structures of two Sm protein complexes and their implications for the assembly of the spliceosomal snRNPs. Cell 96, 375–387 (1999)

    CAS  Article  Google Scholar 

  23. Kastner, B. & Lührmann, R. Electron microscopy of U1 small nuclear ribonucleoprotein particles: shape of the particle and position of the 5′ RNA terminus. EMBO J. 8, 277–286 (1989)

    CAS  Article  Google Scholar 

  24. Kastner, B., Kornstädt, U., Bach, M. & Lührmann, R. Structure of the small nuclear RNP particle U1: identification of the two structural protuberances with RNP-antigens A and 70K. J. Cell Biol. 116, 839–849 (1992)

    CAS  Article  Google Scholar 

  25. Stark, H., Dube, P., Lührmann, R. & Kastner, B. Arrangement of RNA and proteins in the spliceosomal U1 small nuclear ribonucleoprotein particle. Nature 409, 539–542 (2001)

    ADS  CAS  Article  Google Scholar 

  26. Clemons, W. M. et al. Structure of a bacterial 30S ribosomal subunit at 5.5 Å resolution. Nature 400, 833–840 (1999)

    ADS  CAS  Article  Google Scholar 

  27. Ban, N. et al. Placement of protein and RNA structures into a 5 Å-resolution map of the 50S ribosomal subunit. Nature 400, 841–847 (1999)

    ADS  CAS  Article  Google Scholar 

  28. Heinrichs, V., Bach, M., Winkelmann, G. & Lührmann, R. U1-specific protein C needed for efficient complex formation of U1 snRNP with a 5′ splice site. Science 247, 69–72 (1990)

    ADS  CAS  Article  Google Scholar 

  29. Will, C. L. et al. In vitro reconstitution of mammalian U1 snRNPs active in splicing: the U1-C protein enhances the formation of early (E) spliceosomal complexes. Nucleic Acids Res. 24, 4614–4623 (1996)

    CAS  Article  Google Scholar 

  30. Kim, C. H. & Tinoco, I. A retroviral RNA kissing complex containing only two G-C base pairs. Proc. Natl Acad. Sci. USA 97, 9396–9401 (2000)

    ADS  CAS  Article  Google Scholar 

  31. Cowtan, K. D., Zhang, K. Y. J. & Main, P. in International Tables for Crystallography Vol. F: Crystallography of Biological Macromolecules (eds Rossmann, M. G. & Arnold, E.) 705–710 (Kluwer, 2001)

    Google Scholar 

  32. Duckett, D. R., Murchie, A. I. H. & Lilley, D. M. J. The global folding of four-way helical junctions in RNA, including that in U1 snRNA. Cell 83, 1027–1036 (1995)

    CAS  Article  Google Scholar 

  33. Törö, I. et al. RNA binding in an Sm core domain: X-ray structure and functional analysis of an archaeal Sm protein complex. EMBO J. 20, 2293–2303 (2001)

    Article  Google Scholar 

  34. Urlaub, H., Raker, V. A., Kostka, S. & Lührmann, R. Sm protein-Sm site RNA interactions within the inner ring of the spliceosomal snRNP core structure. EMBO J. 20, 187–196 (2001)

    CAS  Article  Google Scholar 

  35. Lu, D., Searles, M. A. & Klug, A. Crystal structure of a zinc-finger-RNA complex reveals two modes of molecular recognition. Nature 426, 96–100 (2003)

    ADS  CAS  Article  Google Scholar 

  36. Urlaub, H., Hartmuth, K., Kostka, S., Grelle, G. & Lührmann, R. A general approach for identification of RNA-protein cross-linking sites within native human spliceosomal small nuclear ribonucleoproteins (snRNPs). J. Biol. Chem. 275, 41458–41468 (2000)

    CAS  Article  Google Scholar 

  37. Zhou, C. & Huang, R. H. Crystallographic snapshots of eukaryotic dimethylallyltransferase acting on tRNA: Insight into tRNA recognition and reaction mechanism. Proc. Natl Acad. Sci. USA 105, 16142–16147 (2008)

    ADS  CAS  Article  Google Scholar 

  38. Du, H. & Rosbash, M. The U1 snRNP protein U1C recognizes the 5′ splice site in the absence of base pairing. Nature 419, 86–90 (2002)

    ADS  CAS  Article  Google Scholar 

  39. Chen, J. Y.-F. et al. Specific alterations of U1-C protein or U1 small nuclear RNA can eliminate the requirement of Prp28p, an essential DEAD box splicing factor. Mol. Cell 7, 227–232 (2001)

    CAS  Article  Google Scholar 

  40. Ismaïli, N., Sha, M., Gustafson, E. H. & Konarska, M. M. The 100-kDa U5 snRNP protein (hPrp28p) contacts the 5′ splice site through its ATPase site. RNA 7, 182–193 (2001)

    Article  Google Scholar 

  41. Steitz, T. A. Visualizing polynucleotide polymerase machines at work. EMBO J. 25, 3458–3468 (2006)

    CAS  Article  Google Scholar 

  42. 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)

    ADS  CAS  Article  Google Scholar 

  43. Mizushima, S. & Nomura, M. Assembly mapping of 30S ribosomal proteins from E. coli . Nature 226, 1214–1218 (1970)

    ADS  CAS  Article  Google Scholar 

  44. Bedford, M. T., Reed, R. & Leder, P. WW domain-mediated interactions reveal a spliceosome-associated protein that binds a third class of proline-rich motif: The proline, glycine and methionine-rich motif. Proc. Natl Acad. Sci. USA 95, 10602–10607 (1998)

    ADS  CAS  Article  Google Scholar 

  45. Dönmez, G., Hartmuth, K., Kastner, B., Will, C. L. & Lührmann, R. The 5′ end of U2 snRNA is in close proximity to U1 and functional sites of the pre-mRNA in early spliceosomal complexes. Mol. Cell 25, 399–411 (2007)

    Article  Google Scholar 

  46. Tazi, J. et al. Thiophosphorylation of U1-70K protein inhibits pre-mRNA splicing. Nature 363, 283–286 (1993)

    ADS  CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  48. Leslie, A. G. W. The integration of macromolecular diffraction data. Acta Crystallogr. D 62, 48–57 (2006)

    Article  Google Scholar 

  49. Evans, P. Scaling and assessment of data quality. Acta Crystallogr. D 62, 72–82 (2006)

    Article  Google Scholar 

  50. Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

  51. de la Fortelle, E. & Bricogne, G. Maximum likelihood heavy-atom parameter refinement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods. Methods Enzymol. 276, 472–493 (1997)

    CAS  Article  Google Scholar 

  52. Knäblein, J. et al. Ta6, a tool for phase determination of large biological assemblies by X-ray crystallography. J. Mol. Biol. 270, 1–7 (1997)

    ADS  Article  Google Scholar 

  53. Abrahams, J. P. & Leslie, A. G. Methods used in the structure determination of bovine mitochondrial F1 ATPase. Acta Crystallogr. D 52, 30–42 (1996)

    CAS  Article  Google Scholar 

  54. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991)

    Article  Google Scholar 

  55. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Article  Google Scholar 

  56. Nagai, K., Oubridge, C., Jessen, T.-H., Li, J. & Evans, P. R. Crystal structure of the RNA-binding domain of the U1 small nuclear ribonucleoprotein A. Nature 348, 515–520 (1990)

    ADS  CAS  Article  Google Scholar 

  57. DeLano, W. L. PyMOL Molecular Viewerhttp://www.pymol.org〉 (2002)

    Google Scholar 

Download references

Acknowledgements

We are grateful to T. Jessen, C. Kambach, J. Avis, R. Young, Y. Muto, S. Walke and T. Ignjatovic for expressing U1 snRNP proteins and laying the foundation of this project. We thank Swiss Light Source, Daresbury and European Synchrotron Radiation Facility beamline staff, particularly C. Schulze-Briese, T. Tomizaki and A. Pauluhn at the Swiss Light Source, for their support; V. Ramakrishnan, A. Newman, A. Andreeva, A. Murzin and C. Vonrhein for discussions; the current members of the Nagai group for help; S. Sengupta for support; and H. Stark for making the cryo-electron microscopy structure available. This project has been funded by the Medical Research Council and the Human Frontier Science Program (HFSP). D.A.P.K. was a recipient of a HFSP long-term fellowship. A.K.W.L was supported by the National Science and Engineering Research Council of Canada, the ORS Fund, the Cambridge Commonwealth Trust and Sidney Sussex College Junior Research Fellowship.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Kiyoshi Nagai.

Supplementary information

Supplementary Information

This file contains Supplementary Table S1 and Supplementary Figures S1 with Legend (PDF 472 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Pomeranz Krummel, D., Oubridge, C., Leung, A. et al. Crystal structure of human spliceosomal U1 snRNP at 5.5 Å resolution. Nature 458, 475–480 (2009). https://doi.org/10.1038/nature07851

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature07851

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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