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CryoEM studies of the spliceosome

Project description

Time-lapsed ‘still shots’ of the spliceosome in action

Gene expression relies on pre-messenger RNA (pre-mRNA) production from nuclear DNA followed by removal of the introns, non-coding regions, and covalent bonding of the extrons, the coding portions, to produce mature mRNA. The latter is the template for protein production in the cytoplasm. The spliceosome is a large RNA-protein complex that catalyses intron removal. Building on its recent determination of the crystal structure of three of five subunits of the spliceosome, the European Research Council-funded SPLICE3D project will determine the structure of the entire spliceosome at different steps of splicing. Putting together these ‘still shots’ will reveal the molecular mechanisms of pre-mRNA splicing and insight into gene expression in health and disease.

Objective

The protein coding sequences of the majority of eukaryotic genes are interrupted by non-coding introns. The spliceosome is an immense and intricate molecular machine that catalyses the excision of introns from pre-mRNAs and splicing together of exons to produce mature mRNA. This is a crucial process in eukaryotic gene expression and we aims to greatly increase our understanding of its molecular mechanism through cryoEM studies of the spliceosome. The spliceosome comprises five canonical subunits, namely U1, U2, U4, U5 and U6 small nuclear ribonucleoprotein particles (snRNPs) and numerous non-snRNP factors. During the initial stages, U1 and U2 snRNPs bind the 5’-splice site and branch point of pre-mRNA, respectively, and the spliceosome is fully assembled when the U4/U6.U5 tri-snRNP is recruited. The spliceosome then becomes activated through extensive structural and compositional remodeling which leads to the formation of the catalytic RNA core. Recently we determined the crystal structure of U1 snRNP and two key spliceosomal proteins: Prp8, which harbours the catalytic RNA core and Brr2, which catalyses the rearrangement of the RNA interaction network and plays a crucial role in spliceosomal activation. We also solved the structure of U4/U6.U5 tri-snRNP by cryoEM and revealed the nearly complete organisation of U5 snRNA and U4/U6 snRNA and over 30 proteins within this complex, providing crucial insights into the activation mechanism and the active site of the spliceosome. Building on these achievements we aim to determine the structure of the entire spliceosome stalled at different steps of the splicing reaction so that the molecular mechanism of pre-mRNA splicing is understood in structural terms. This will enormously increase our understanding of this fundamental process of eukaryotic gene expression and such knowledge will clarify the molecular pathology of diseases caused by mutations in spliceosomal components and may facilitate therapeutic intervention.

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Topic(s)

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Funding Scheme

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ERC-ADG - Advanced Grant

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Call for proposal

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(opens in new window) ERC-2015-AdG

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Host institution

UNITED KINGDOM RESEARCH AND INNOVATION
Net EU contribution

Net EU financial contribution. The sum of money that the participant receives, deducted by the EU contribution to its linked third party. It considers the distribution of the EU financial contribution between direct beneficiaries of the project and other types of participants, like third-party participants.

€ 1 069 584,70
Address
POLARIS HOUSE NORTH STAR AVENUE
SN2 1FL SWINDON
United Kingdom

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Region
South West (England) Gloucestershire, Wiltshire and Bristol/Bath area Swindon
Activity type
Research Organisations
Links
Total cost

The total costs incurred by this organisation to participate in the project, including direct and indirect costs. This amount is a subset of the overall project budget.

€ 1 069 584,70

Beneficiaries (1)

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