Project Code D7
Molecular and structural mechanisms for accurate 3’-splice site selection during mRNA synthesis by the human spliceosome
Mammalian genes are transcribed into precursor messenger RNAs (pre-mRNAs), from which the spliceosome excises non-coding introns in the nucleus before the mRNA is exported to the cytoplasm and translated into proteins.
The spliceosome is a dynamic RNA-protein complex that assembles the novo on each pre-mRNA and catalyses two sequential reactions – branching and exon ligation – to produce mRNAs with continuous protein-coding information. Following branching, the ATPase Prp16 remodels the spliceosome to promotes docking of the 3’ splice site (3’-SS) in the active site, where it is stabilised by specific exon ligation factors. After exon ligation, the ATPase Prp22 releases the mature mRNA for export to the cytoplasm. Importantly, Prp22 also proofreads docking of the 3’-SS to ensure correct mRNA synthesis1.
Electron cryo-microscopy (cryo-EM) has revealed the molecular structures of the branching and exon ligation conformations of the spliceosome, showing how splice sites are recognised and how specific factors stabilize each catalytic conformation2,3. However, the molecular mechanisms that promote accurate 3’-SS selection and during exon ligation remain poorly understood, especially in humans, where accurate mRNA synthesis must be balanced with regulated alternative 3’-SS use.
Recently, using an in vitro yeast system, we have uncovered additional intermediates during Prp16 remodelling, suggesting there are unexplored dynamics during selection and docking of 3’-SS in the active site4. Indeed, recent cryo-EM studies revealed that several novel human exon ligation factors promote mRNA formation2,3. These include factors misfolded in neurodegenerative disease such as FAM50A, as well as proteins downregulated (FAM32A), or mutated (DDX41, NKAP), in specific cancers. These factors are recruited to the spliceosome during Prp16-mediated remodelling and modulate alternative splicing. Some of these factors (e.g. FAM32A) promote docking of the 3’-SS in the active site, while others (e.g. FAM50A) may specifically modulate the proofreading activity of Prp22 to ensure correct mRNA synthesis.
These novel exon ligation factors may bind the spliceosome sequentially and act as chaperones to modulate fidelity of 3’-SS selection by forming short-lived, transient spliceosome intermediates. To understand how these factors modulate spliceosome dynamics and to capture transient intermediates, we aim to develop an in vitro system for the catalytic stage of splicing and study spliceosome remodelling during catalysis using time-resolved cryo-EM. We aim to establish a vitrification set-up that combines time control of the reaction with spray-based grid deposition (currently under development at eBIC) and assessment of complex composition using mass photometry, for which we are developing a collaboration with Philipp Kukura’s group in Chemistry. As a dynamic complex remodelled by ATP, the spliceosome offers an ideal sample for the development of time-resolved cryo-EM methods to access transient states that may be in thermodynamic equilibrium and thus inaccessible to conventional cryo-EM sample preparation methods. This work would unravel the molecular mechanisms by which several disease-associated splicing factors modulate correct mRNA synthesis and alternative splicing during splicing catalysis.
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., Oubridge, C., Wilkinson, M. E., Newman, A. J. & Nagai, K. A human postcatalytic spliceosome structure reveals essential roles of metazoan factors for exon ligation. Science (New York, NY) 363, 710–714 (2019).
3. Dybkov, O. et al. Regulation of 3′ splice site selection after step 1 of splicing by spliceosomal C* proteins. Sci Adv 9, eadf1785 (2023).
4. Wilkinson, M. E., Fica, S. M., Galej, W. P. & Nagai, K. Structural basis for conformational equilibrium of the catalytic spliceosome. Mol Cell (2021) doi:10.1016/j.molcel.2021.02.021.
For more information about the Fica lab see: https://snrnpsplicingbiochemlab.web.ox.ac.uk/
Dr Sebastian Fica | Biochemistry (ox.ac.uk)
For informal enquiries: Sebastian.email@example.com