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.
Field of science
- /natural sciences/biological sciences/genetics and heredity/rna
- /natural sciences/biological sciences/biochemistry/biomolecules/proteins/proteomics
- /natural sciences/chemical sciences/analytical chemistry/mass spectrometry
Call for proposal
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