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Structural and functional characterization of the core splicing factor PRP8 Open Access


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Type of item
Degree grantor
University of Alberta
Author or creator
Mehta, Garima
Supervisor and department
MacMillan, Andrew (Biochemistry)
Examining committee member and department
LaPointe, P (Biochemistry)
Lemieux, J (Biochemistry)
Glover, M (Biochemistry)
Department of Biochemistry

Date accepted
Graduation date
Master of Science
Degree level
Over 90% of eukaryotic genes are initially expressed as precursor-messenger RNAs (pre-mRNAs) that contain coding exon sequences interrupted by non-coding intron sequences. The introns are excised and exons are ligated together to form messenger RNA (mRNA) through a process called splicing. Splicing involves two sequential transesterification steps catalyzed by a large RNA/protein complex called the spliceosome. A number of studies implicate a protein called Prp8 at the heart of the catalytic core of the spliceosome. I am interested in determining the exact role of the RNase H domain of Prp8 in regulating the individual steps of splicing and the transition between the two steps of splicing. In order to understand the function of Prp8 in this regard, I have created panels of mutant RNase H Prp8 proteins that are predicted to affect either the switch between Prp8 first step and second step conformations or the ability of Prp8 to bind a metal ion in the second step conformation. The human Prp8 mutant alleles will be characterized structurally by X-ray methods and the corresponding yeast Prp8 alleles will be tested for their splicing activity both within yeast and biochemical extracts prepared from yeast. In particular, our model suggests switch dependent interactions of the RNase H domain with the rest of Prp8 and these experiments will test this. The experiments with respect to metal binding are important because they will provide more definitive information regarding the role of Prp8 in the chemistry of the splicing reaction. These results will be of great interest because it has long been believed that RNA, rather than protein components, of the spliceosome were solely responsible for promoting the splicing chemistry.
Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
Citation for previous publication
Ritchie, D.B., Schellenberg, M.J., Gesner, E.M., Raithatha, S.A., Stuart, D.T., and MacMillan, A.M. (2008). Structural elucidation of a PRP8 core domain from the heart of the spliceosome. Nat. Struct. Mol. Biol. 15, 1199-1205.

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