Structural and functional studies of the core splicing factor Prp8

  • Author / Creator
    Wu, Tao
  • More than 90% of human genes undergo a processing step called splicing, whereby noncoding introns are removed from initial transcripts and coding exons are ligated together to yield mature messenger RNA. Splicing involves two sequential transesterification reactions catalyzed by a large RNA/protein complex called the spliceosome. RNA components of the spliceosome are at least partly responsible for splicing catalysis. However, the precise architecture of the spliceosome active site and whether it includes protein components remain unknown. Numerous genetic and biochemical analyses have placed one of the largest and most highly conserved of nuclear proteins, Prp8, at the heart of the catalytic core of the splicing machinery during spliceosome assembly and through catalysis. Here we provide structural and functional evidence that the RNase H domain of Prp8 undergoes a conformational change during splicing which unmasks a metal-binding site required for the second step of splicing. We are able to demonstrate that a magnesium ion essential for the catalysis binds to the RNase H domain of Prp8 and a metal specificity switch which abrogates metal binding severely inhibits the second step of splicing. We also show that yeast prp8 first- and second-step alleles correspond to Prp8 mutants that favour one of the two distinct Prp8 conformations observed in the crystal structure of Prp8 RNase H domain. Together these data support the model of rearrangements within the spliceosome at the time of transition between the first and second step of splicing. Our findings also establish that Prp8 is a metalloprotein which promotes exon ligation and are consistent with the designation of the spliceosome as a ribonucleoprotein enzyme.

  • Subjects / Keywords
  • Graduation date
    Spring 2012
  • 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
    • Kothe, Ute (Chemistry and Biochemistry, University of Lethbridge)
    • Glover, Mark J.N. (Biochemistry)
    • Owttrim, George (Biological Sciences)
    • MacMillan, Andrew (Biochemistry)
    • Schultz, Michael C. (Biochemistry)