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Splicing, Prp8

Mehta, Garima

Supervisor and department: MacMillan, Andrew Biochemistry

Examining committee member and department: LaPointe, P Biochemistry Lemieux, J Biochemistry Glover, M Biochemistry

Department: Department of Biochemistry

Specialization:

Date accepted: 2015-05-08T15:33:34Z

Graduation date: 2015-11

Degree: Master of Science

Degree level: Master's

Abstract: 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.

Language: English

DOI: doi:10.7939-R3TM7260P

Rights: 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.





Author: Mehta, Garima

Source: https://era.library.ualberta.ca/


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Structural and functional characterization of the core splicing factor PRP8 by Garima Mehta A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science Department of Biochemistry University of Alberta © Garima Mehta, 2015 Abstract: 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 ii because it has long been believed that RNA, rather than protein components, of the spliceosome were solely responsible for promo...





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