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).
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 (Galej et al., 2013), Brr2 (Nguyen et al., 2013) and Snu114. It provided important insight into the active site and activation mechanism of the spliceosome.
Recently we have solved a high resolution cryo-EM structure of the whole spliceosome captured immediately after the first step of catalysis (Galej et al., 2016). We have built an atomic model for this nearly 2MDa complex composed of 44 subunits including proteins and RNAs. Most importantly, the data allowed them to visualise for the very first time the pre-mRNA substrate inside the active spliceosome and its interactions with the RNA catalytic core and surrounding proteins. This has provided important mechanistic insights into RNA-based catalysis. It has consolidated nearly three decades of biochemistry and genetics on the spliceosome and indicates new research directions to be explored.
Our work provides crucial insights into the structure and function of the spliceosome as well as the evolutionary origin of the spliceosome.
- Galej, W. P., Wilkinson, M. E., Fica, S. M., Oubridge, C., Newman, A. J. and Nagai, K. (2016)
CryoEM structure of the spliceosome immediately after branching
Nature, DOI: 10.1038/nature19316
- 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
- Andrew Newman
- Pei-chun Lin
- Sebastian Fica
- Lisa Strittmatter
- Max Wilkinson
- Clemens Plaschka