STRUCTUREU4U6SNRNP

Structural and Biochemical Examination of the Yeast U4/U6 snRNP

Coordinatore	MEDICAL RESEARCH COUNCIL    more  Organization address address: NORTH STAR AVENUE POLARIS HOUSE city: SWINDON postcode: SN2 1FL contact info Titolo: Ms. Nome: Elizabeth Cognome: Cutler Email: send email Telefono: +44 122 340 2357 Fax: +44 122 341 2515
Nazionalità Coordinatore	United Kingdom [UK]
Totale costo	201˙049 €
EC contributo	201˙049 €
Programma	FP7-PEOPLE Specific programme "People" implementing the Seventh Framework Programme of the European Community for research, technological development and demonstration activities (2007 to 2013)
Code Call	FP7-PEOPLE-2010-IIF
Funding Scheme	MC-IIF
Anno di inizio	2011
Periodo (anno-mese-giorno)	2011-04-01   -   2013-03-31

 Partecipanti

#	participant	country	role	EC contrib. [€]
1	MEDICAL RESEARCH COUNCIL  Organization address address: NORTH STAR AVENUE POLARIS HOUSE city: SWINDON postcode: SN2 1FL contact info Titolo: Ms. Nome: Elizabeth Cognome: Cutler Email: send email Telefono: +44 122 340 2357 Fax: +44 122 341 2515	UK (SWINDON)	coordinator	201˙049.60

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 Obiettivo del progetto (Objective)

'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.'