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.
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References
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)
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)
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)
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)
Jurica, M. S. & Moore, M. J. Pre-mRNA splicing: awash in a sea of proteins. Mol. Cell 12, 5–14 (2003)
Makarov, E. M. et al. Small nuclear ribonucleoprotein remodeling during catalytic activation of the spliceosome. Science 298, 2205–2208 (2002)
Guthrie, C. & Patterson, B. Spliceosomal snRNAs. Annu. Rev. Genet. 22, 387–419 (1988)
Griffiths-Jones, S. et al. Rfam: annotating non-coding RNAs in complete genomes. Nucleic Acids Res. 33, D121–D124 (2005)
Zhuang, Y. & Weiner, A. M. A compensatory base change in U1 snRNA suppresses a 5′ splice site mutation. Cell 46, 827–835 (1986)
Ruby, S. W. & Abelson, J. An early hierarchic role of U1 small nuclear ribonucleoprotein in spliceosome assembly. Science 242, 1028–1035 (1988)
Séraphin, B. & Rosbash, M. Identification of functional U1 snRNA-pre-mRNA complexes committed to spliceosome assembly and splicing. Cell 59, 349–358 (1989)
Kohtz, J. D. et al. Protein–protein interactions and 5′-splice-site recognition in mammalian mRNA precursors. Nature 368, 119–124 (1994)
Graveley, B. R. Sorting out the complexity of SR protein functions. RNA 6, 1197–1211 (2000)
Krol, A. et al. Solution structure of human U1 snRNA. Derivation of a possible three-dimensional model. Nucleic Acids Res. 18, 3803–3811 (1990)
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)
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)
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)
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)
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)
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)
Muto, Y. et al. The structure and biochemical properties of the human spliceosomal protein U1C. J. Mol. Biol. 341, 185–198 (2004)
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)
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)
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)
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)
Clemons, W. M. et al. Structure of a bacterial 30S ribosomal subunit at 5.5 Å resolution. Nature 400, 833–840 (1999)
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)
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)
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)
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)
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)
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)
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)
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)
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)
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)
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)
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)
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)
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)
Steitz, T. A. Visualizing polynucleotide polymerase machines at work. EMBO J. 25, 3458–3468 (2006)
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)
Mizushima, S. & Nomura, M. Assembly mapping of 30S ribosomal proteins from E. coli . Nature 226, 1214–1218 (1970)
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)
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)
Tazi, J. et al. Thiophosphorylation of U1-70K protein inhibits pre-mRNA splicing. Nature 363, 283–286 (1993)
Smith, C. W. & Valcárcel, J. Alternative pre-mRNA splicing: the logic of combinatorial control. Trends Biochem. Sci. 25, 381–388 (2000)
Leslie, A. G. W. The integration of macromolecular diffraction data. Acta Crystallogr. D 62, 48–57 (2006)
Evans, P. Scaling and assessment of data quality. Acta Crystallogr. D 62, 72–82 (2006)
Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)
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)
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)
Abrahams, J. P. & Leslie, A. G. Methods used in the structure determination of bovine mitochondrial F1 ATPase. Acta Crystallogr. D 52, 30–42 (1996)
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)
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)
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)
DeLano, W. L. PyMOL Molecular Viewer 〈http://www.pymol.org〉 (2002)
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.
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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
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DOI: https://doi.org/10.1038/nature07851
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