The removal of introns from nuclear pre-mRNA and splicing together of exons into a continuous translatable coding sequence is catalyzed by a large and dynamic RNA-protein machine known as the spliceosome. Five small nuclear ribonucleoprotein particles (U1, U2, U4/U6 and U5 snRNPs) and numerous non-snRNP factors assemble at each intron in the pre-mRNA to form a spliceosome. Subsequently the spliceosome undergoes extensive compositional and structural changes to become catalytically active. How does the spliceosome assemble and carry out its function? How did a molecular machine as immense and complex as the spliceosome evolve in the Eukaryotic lineage? Our project aims to answer these questions by solving structures of the whole spliceosome and its key components by crystallography and electron cryo-microscopy (cryoEM).
U1 snRNP binds to the 5’ splice site of pre-mRNA and initiates the assembly of the spliceosome. The human spliceosomal U1 snRNP consists of U1 small nuclear RNA (snRNA) and 10 proteins. We have determined the crystal structure of the functional core of human U1 snRNP revealing the architectural principle of the spliceosomal snRNPs and the structural basis of 5'-splice site recognition (Pomeranz Krummel et al., 2009; Kondo et al., 2015).
Recently we determined the structure of yeast U4/U6.U5 tri-snRNP by cryoEM single-particle reconstruction first at 5.9 Å resolution (Nguyen et al., 2015) and then at 3.7 Å overall resolution (Nguyen et al., 2016). U4/U6.U5 tri-snRNP is a 1.5-megadalton pre-assembled spliceosomal complex, which represents a substantial part of the spliceosome prior to activation. We have been able to build a near complete atomic model of this complex comprising U5 snRNA, U4 snRNA, U6 snRNA and more than 30 proteins, including the key components Prp8, Brr2 and Snu114. It provided important insight into the active site and activation mechanism of the spliceosome.
Structural and mechanistic similarities between the spliceosome’s RNA core and Group II self-splicing introns argue that these splicing machines very likely have a common ancestry and that the spliceosomal snRNAs are fragments of group II intron. We showed that the large domain of Prp8 has a similar domain architecture to Group II intron encoded proteins, providing the first experimental evidence that a protein component of the spliceosome and Group II intron encoded protein have a common evolutionary origin (Galej et al., 2013). The structure of U4/U6.U5 tri-snRNP shows that the large domain of Prp8 is located at the centre of the assembly and, together with additional domains recruited later, functions as a hub of protein–protein and protein–RNA interactions to facilitate the reassembly of group II intron-like ribozyme from the spliceosomal snRNAs.
Our work provides crucial insights into the structure and function of the spliceosome as well as the evolutionary origin of the spliceosome.
- Nguyen, T.H.D., Galej, W.P., Bai, X-C., Oubridge, C., Newman, A.J., Scheres, S.H.W. and Nagai, K. (2016)
Cryo-EM structure of the yeast U4/U6.U5 tri-snRNP at 3.7 Å resolution
Nature 530: 298-302
- Nguyen, T.H.D., Galej, W.P., Bai, X-C., Savva, C.G., Newman, A.J., Scheres, S.H.W. and Nagai, K. (2015)
The architecture of the spliceosomal U4/U6.U5 tri-snRNP
Nature 523: 47-52
- Kondo, Y., Oubridge, C., van Roon, A.M. and Nagai, K. (2015)
Crystal structure of human U1 snRNP, a small nuclear ribonucleoprotein particle, reveals the mechanism of 5' splice site recognition
Elife 4: e04986
- Nguyen, T.H., Li, J., Galej, W.P., Oshikane, H., Newman, A.J. and Nagai, K. (2013)
Structural basis of Brr2-Prp8 interactions and implications for U5 snRNP biogenesis and the spliceosome active site
Structure 21: 910-919.
- Galej, W.P., Oubridge, C., Newman, A.J. and Nagai, K. (2013)
Crystal structure of Prp8 reveals active site cavity of the spliceosome
Nature 493: 638-643.
- Leung, A.K.W., Nagai, K. and Li, J. (2011)
Structure of the spliceosomal U4 snRNP core domain and its implication for snRNP biogenesis
Nature 473: 536-539.
- Pomeranz Krummel, D.A., Oubridge, C., Leung, A.K.W., Li, J. and Nagai, K. (2009)
Crystal structure of human spliceosomal U1 snRNP at 5.5 Å resolution.
Nature 458: 475-480.
- Christine Norman
- Chris Oubridge
- Wojciech Galej
- Andrew Newman
- Pei-chun Lin
- Sebastian Fica
- Lisa Strittmatter
- Song Tan
- Max Wilkinson
- Clemens Plaschka