Structural and biochemical analysis of the essential spliceosomal protein Prp8

  • Author / Creator
    Ritchie, Dustin B.
  • More than 90% of human genes undergo a processing step called splicing, whereby non-coding introns are removed from initial transcripts and coding exons are ligated together to yield mature messenger RNA. Roughly 50% of human genetic diseases correspond to aberrant splicing. Splicing is catalyzed by an RNA/protein machine called the spliceosome. RNA components of the spliceosome are at least partly responsible for splicing catalysis. In addition, in vitro analyses implicate an essential and very highly conserved protein, Prp8, in orchestrating key steps in spliceosome assembly and possibly catalysis. Interestingly, mutant alleles of Prp8 are the cause of retinitis pigmentosa, an inherited form of retinal degeneration.
    A key goal is elucidation of the precise role of Prp8 in the spliceosome by high resolution structural analysis. The large size of Prp8 and its insolubility hinder progress in this regard. Instead, structural understanding of Prp8 can be gained by investigating domains in isolation; however there is only limited information as to what domain boundaries are and few hints about the functional relevance of putative domains. Here we have further defined the previously proposed domain IV in Prp8, and identified the domain IV core. Structural determination of the domain IV core reveals an RNase H fold, which could not be predicted based on primary sequence alone. RNase H recognizes A-form nucleic acid duplexes, which strongly suggests the domain IV core interacts with double-stranded RNA in the context of the spliceosome. Characterizing the binding preferences of the domain IV revealed the highest affinity is for a 4-helix junction structure adopted by the very RNAs at the spliceosome active site. Our characterization of the protein/RNA binding interface by complementary footprinting techniques currently provides the best model of how RNA interacts with an essential protein component at the heart of the spliceosome.

  • Subjects / Keywords
  • Graduation date
    Spring 2010
  • Type of Item
  • Degree
    Doctor of Philosophy
  • 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
  • Supervisor / co-supervisor and their department(s)
  • Examining committee members and their departments
    • Glover, Mark (Biochemistry)
    • Owttrim, George (Biological Sciences)
    • Chabot, Benoit (Microbiology & Infectious Diseases, University of Sherbrooke)
    • Fahlman, Richard (Biochemistry)