Structural and functional characterization of the core splicing factor PRP8

  • Author / Creator
    Mehta, Garima
  • 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.

  • Subjects / Keywords
  • Graduation date
  • Type of Item
  • Degree
    Master of Science
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
  • Institution
    University of Alberta
  • Degree level
  • Department
    • Department of Biochemistry
  • Supervisor / co-supervisor and their department(s)
    • MacMillan, Andrew (Biochemistry)
  • Examining committee members and their departments
    • Lemieux, J (Biochemistry)
    • LaPointe, P (Biochemistry)
    • Glover, M (Biochemistry)