Project code M5
Molecular mechanisms underlying the function of ATPases involved in proofreading and disassembly of pre-mRNA splicing by the human spliceosome
Mammalian genes are transcribed into precursor messenger RNAs (pre-mRNAs), from which non-coding introns are spliced out in the nucleus before the mRNA is exported to the cytoplasm and translated into proteins. Introns expand proteomic diversity by allowing a single gene to encode multiple mRNA isoforms, coding for multiple protein isoforms with distinct activities. Introns are excised by the spliceosome – a dynamic assembly of RNA and proteins and splicing errors are implicated in up to 30% of human diseases.
The spliceosome assembles de novo on each pre-mRNA and catalyses two sequential transesterifications at a single RNA-based active site to excise a specific intron and ligate the flanking exons into mRNA 1. During catalysis, several trans-acting ATPases modulate the transitions between different conformations spliceosome. These ATPases promote the exchange of reactants at the active site, allow exchange of protein factors that stabilize each conformation, and proofread fidelity of splice site choice2. Following mRNA synthesis, the mRNA is released by the action of the ATPase Prp22, while the ATPase Prp43 disassembles the resulting intron-lariat spliceosome (ILS) to release the excised intron for degradation.
In the last six years, structures of different conformation of the yeast spliceosome have provided a molecular view of the basic mechanism of splicing in yeast, showing how the splice sites are recognised and how specific factors stabilize each catalytic conformation2. The structure of the yeast post-catalytic spliceosome (P complex) suggested a mechanism by which Prp22 releases the mRNA, while the structure of the yeast ILS provided some structural insights into disassembly of the spliceosome by Prp43 and its associated co-factors3. Importantly, in yeast, proofreading of correct splice site usage by Prp22 is coupled to a discard pathway in which Prp43 disassembles spliceosomes that utilise incorrect splice sites and are rejected by Prp224,5 . It remains unclear where Prp43 binds in the human ILS6 and it is not known what specific RNA component of the spliceosome is targeted by Prp43 during discard and disassembly in mammals.
Although the active site and basic splice site recognition is conserved from yeast to humans, several additional ATPases associate with the human spliceosome and have been implicated in splicing fidelity7,8. Indeed, in mammals fidelity of splice site choice often relies on recognition of only 1-2 nucleotides around highly variable splice sites, which must be balanced with alternative splicing. Thus, how Prp22, Prp43, and other mammalian ATPases act to safeguard splicing fidelity remains poorly understood.
We aim to establish new biochemical systems in vitro, and potentially in vivo, to study proofreading of splice site use and spliceosome disassembly in humans. Our goals are to identify the specific complexes involved in these processes and to use this system to trap intermediates during proofreading, discard, and disassembly. Electron cryomicroscopy will then be employed to obtain molecular insights into the mechanism of action of Prp22, Prp43, and associated co-factors. These structural studies will be complemented by biochemical assays and new CLIP RNA crosslinking and sequencing approaches9 to elucidate the mechanisms underlying proofreading of correct splice site choice.
1. Wilkinson, M. E., Charenton, C. & Nagai, K. RNA Splicing by the Spliceosome. Annual Review of Biochemistry 89, 1–30 (2019).
2. Fica, S. M. & Nagai, K. Cryo-electron microscopy snapshots of the spliceosome: structural insights into a dynamic ribonucleoprotein machine. Nature structural & molecular biology 24, 791–799 (2017).
3. Wan, R., Yan, C., Bai, R., Lei, J. & Shi, Y. Structure of an Intron Lariat Spliceosome from Saccharomyces cerevisiae. Cell 171, 120-132.e12 (2017).
4. Mayas, R. M., Maita, H., Semlow, D. R. & Staley, J. P. Spliceosome discards intermediates via the DEAH box ATPase Prp43p. Proceedings of the National Academy of Sciences 107, 10020–10025 (2010).
5. Mayas, R. M., Maita, H. & Staley, J. P. Exon ligation is proofread by the DExD/H-box ATPase Prp22p. Nature structural & molecular biology 13, 482–490 (2006).
6. Zhang, X. et al. Structures of the human spliceosomes before and after release of the ligated exon. Cell Research 29, 1–285 (2019).
7. Fica, S. M. Cryo-EM snapshots of the human spliceosome reveal structural adaptions for splicing regulation. Current Opinion in Structural Biology 65, 139–148 (2020).
8. Sales-Lee, J. et al. Coupling of spliceosome complexity to intron diversity. Biorxiv 2021.03.19.436190 (2021) doi:10.1101/2021.03.19.436190.
9. Strittmatter, L. M. et al. psiCLIP reveals dynamic RNA binding by DEAH-box helicases before and after exon ligation. Nat Commun 12, 1488 (2021).
For more information about the Fica lab see https://snrnpsplicingbiochemlab.web.ox.ac.uk
Dr Sebastian Fica | Biochemistry (ox.ac.uk)
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