Investigating 3´ Splice Site Recognition by the Pre-Spliceosome

• Author / Creator

  McDonald, Jeffrey

• Eukaryotes feature a split gene structure in which pre-mRNAs are spliced into mature RNAs prior to the translation of proteins. The splicing process is catalyzed by a dynamic multi-subunit molecular machine known as the spliceosome that assembles in a stepwise fashion. An early complex formed in the spliceosome assembly pathway is the pre-spliceosome, otherwise referred to as the A complex.The A complex factors SF3B1, SF3B6 and U2AF play a role in the recognition of important consensus sequences at the 3´ splice sites of pre-mRNA introns. SF3B1 and SF3B6 interact about the branch point and the U2AF heterodimer binds the polypyrimidine tract and 3´ splice site. The molecular mechanism of branch point and splice site selection by these factors remains unclear. SF3B6 has been biochemically suggested to directly bind the branch point adenosine, yet structural characterizations of spliceosomes have placed it in a position peripheral to this adenosine. These observed positional inconsistencies may reflect the dynamic nature of splicing and spliceosome assembly. And although essential in humans, SF3B6 may be deleted from the model organism Schizosaccharomyces pombe to yield a phenotypically distinct strain. To carry out splicing in SF3B6 knockout cells, spliceosomes must be assembling in the absence of SF3B6.This thesis aimed to investigate the molecular mechanism of branch point and 3´ splice site selection by SF3B1, SF3B6 and U2AF in the pre-spliceosome. A series of SF3B1-SF3B6 and U2AF expression constructs were analyzed for the in vitro reconstitution of a ternary complex suitable for structural characterization and pre-mRNA binding analysis. Furthermore, biochemical characterization of the A complex in this thesis has demonstrated the formation of an SF3B6 deficient A complex. Preliminary work has also been conducted towards an affinity purification of the A complex and toward the site-specific derivatization of SF3B6 to chemically map pre-spliceosome RNA and protein contacts.

• Subjects / Keywords

  • Splicing
  • SF3B
  • U2AF
  • splice site

• Graduation date

  Spring 2022

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-nyy8-v520

• 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.