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Cryo-EM structural analysis of small nuclear ribonucleoprotein particles (snRNP) biogenesis

Project description

Structural studies of spliceosome biogenesis

Eukaryotic genes are transcribed into pre-mRNAs, which subsequently undergo a process known as splicing to produce mature mRNA. Splicing is carried out by a dynamic multi-subunit machine called the spliceosome. The scope of the EU-funded StructuRNP project is to investigate the biogenesis of the building blocks of the spliceosome, the so-called snRNPs (small nuclear ribonucleoproteins). Researchers will employ biochemistry, cryo-electron microscopy and cross-linking mass spectrometry to determine the structures of the participating proteins and their role in spliceosome biogenesis. The expected results will reveal how regulated assembly of the spliceosome occurs.

Objective

The spliceosome is a multi-megadalton enzyme that catalyses the excision of non-coding introns from nuclear pre-mRNA. It is assembled from five small nuclear ribonucleoprotein particles (snRNPs) and non-snRNP factors, while each snRNP contains a unique small nuclear RNA (snRNA, U1-U5) and is bound by the heptameric Sm-ring. Acquisition of the Sm-ring is a key quality control step in snRNP biogenesis, defects of which are linked with several human diseases. In humans, snRNP biogenesis requires at least 15 proteins, including the 9-subunit survival of motor neuron (SMN) complex. The SMN complex carries out the regulated and specific loading of the Sm ring onto snRNA. Recent studies have revealed the structure of several key proteins that play a role in snRNP assembly, however the complete structures of either the SMN complex alone or with interacting Sm ring and or the snRNP as a whole, are lacking and culminate to a big gap in our knowledge of the snRNP assembly. In this proposal, I will reconstitute key steps in snRNP biogenesis in humans and will determine their structures using cryo-electron microscopy and cross-linking mass spectrometry. I will study how the SMN complex specifically loads the Sm-ring onto U1 snRNA, as a model snRNP. Additionally, purification and structural analysis of endogenous SMN complex will reveal SMN complex stoichiometry and validate the in vitro work. The structures will reveal the interacting partners and the structural transitions within the assembled snRNP and will enable structure-guided biochemical and tissue culture validation of SMN complex function, while my background in protein biochemistry and human tissue culture makes me ideally suited to carry out this high-impact project. StructuRNP will provide first insights into the molecular events and choreography of the spliceosome, providing a leap in our understanding of spliceosome function and thus will have far reaching implications for RNA and spliceosome fields.

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Programme(s)

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Topic(s)

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Funding Scheme

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MSCA-IF - Marie Skłodowska-Curie Individual Fellowships (IF)

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Call for proposal

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(opens in new window) H2020-MSCA-IF-2020

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Coordinator

FORSCHUNGSINSTITUT FUR MOLEKULARE PATHOLOGIE GESELLSCHAFT MBH
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.

€ 186 167,04
Address
CAMPUS-VIENNA-BIOCENTER 1
1030 Wien
Austria

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Region
Ostösterreich Wien Wien
Activity type
Private for-profit entities (excluding Higher or Secondary Education Establishments)
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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.

€ 186 167,04
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