Structural and Biochemical Examination of the Yeast U4/U6 snRNP.

Informations projet

STRUCTUREU4U6SNRNP

N° de convention de subvention: 273372

Projet clôturé

Date de début 1 Avril 2011

Date de fin 31 Mars 2013

Financé au titre de

• Specific programme "People" implementing the Seventh Framework Programme of the European Community for research, technological development and demonstration activities (2007 to 2013)

Coût total € 201 049,60

Contribution de l’UE € 201 049,60

Coordonné par

MEDICAL RESEARCH COUNCIL
Royaume-Uni

Objectif

The spliceosome is a large RNA–protein assembly that catalyzes the removal of introns from mRNA precursors (pre-mRNA) and the splicing of coding exons to produce mature mRNAs. Four small nuclear ribonucleoprotein particles (U1, U2, U5 and U4/U6 snRNPs) assemble onto pre-mRNA substrates together with non-snRNP proteins to form the spliceosome. After binding of the U1 snRNP and the U2 snRNP to the 5’ splice site and the branch point sequence within the intron, a pre-assembled U4/U6.U5 tri-snRNP joins and the spliceosome undergoes extensive remodeling to yield the catalytically active spliceosome. In the tri-snRNP, the U4 and U6 snRNAs are extensively base-paired but upon activation this duplex is unwound by an RNA helicase, Brr2p. The ultimate aim of this project is to determine the structure of the U4/U6 snRNP by X-ray crystallography and reveal details of spliceosome activation/mechanism. This work will initially employ electrophoretic gel mobility shift assays and gel filtration chromatography for characterization of assembly, isothermal titration calorimetry to identify/quantify protein-protein interactions, chemical/enzymatic RNA footprinting to identify protein-RNA contacts and protein-induced conformational changes in the RNA, and pulse-chase quantitative mass spectrometry to further substantiate and characterize the pathway of assembly of the U4/U6 snRNP and the U4/U6.U5 tri-snRNP biochemically and thermodynamically. These experiments will also aid crystallization. Unwinding directionality and helicase-induced protein displacement by the U5 snRNP protein Brr2p will be examined in the context of the fully assembled U4/U6 snRNP and tri-snRNP complexes. This will reveal details of spliceosome activation/mechanism. The proposed project will exploit my extensive knowledge and experience with RNA/protein complex biochemistry, complex engineering skills, and crystallographic approaches but will also allow me to develop new knowledge and skills.

Champ scientifique (EuroSciVoc)

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• sciences naturelles › sciences biologiques › biochimie › biomolécule › protéines › protéomique
• sciences naturelles › sciences de la Terre et sciences connexes de l'environnement › géologie › minéralogie › cristallographie
• sciences naturelles › sciences chimiques › chimie analytique › calorimétrie
• sciences naturelles › sciences biologiques › génétique › ARN
• sciences naturelles › sciences chimiques › chimie analytique › spectrométrie de masse

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Programme(s)

• FP7-PEOPLE - Specific programme "People" implementing the Seventh Framework Programme of the European Community for research, technological development and demonstration activities (2007 to 2013)

Thème(s)

• FP7-PEOPLE-2010-IIF - Marie Curie Action: "International Incoming Fellowships"

Appel à propositions

FP7-PEOPLE-2010-IIF
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Régime de financement

MC-IIF -

Coordinateur

MEDICAL RESEARCH COUNCIL

• Contribution de l’UE: € 201 049,60
• Adresse: 20 Park Crescent ; W1B 1AL LONDON ; Royaume-Uni
• Type d’activité: Organismes publics (à l’exclusion des organismes de recherche et des établissements d’enseignement secondaire ou supérieur)
• Coût total: Aucune donnée