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Intron Recognition at the 3' Splice Site by U2AF, SF1 and p14
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- Author / Creator
- Grewal, Charnpal
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The discontinuous split-gene structure is a core feature of eukaryotic gene architecture and requires pre-mRNA splicing to remove silent intron sequences from the initial pre-mRNA transcript before it becomes a mature mRNA suitable for protein synthesis. Splicing begins with recognition of both the 5′ and 3′ SS (splice site) of the intron, and the 3′ SS is recognized by the heterotrimeric U2AF/SF1 protein complex.
Here, U2AF dimer and U2AF/SF1 trimer variants were expressed from the fission yeast, Schizosaccharomyces pombe. SEC-MALLS and spectroscopy established that the free and RNA-bound complexes are stable, soluble, and monodisperse. This was followed by EMSAs (electrophoretic mobility shift assays) revealing that broadly, SF1 has a major role in binding the 3′ SS and modulates sequence specificity and affinity of the U2AF/SF1 complex for the 3′ SS. Structural characterization via SAXS revealed that U2AF dimer and U2AF/SF1 trimer are well folded and roughly globular in both free and RNA-bound states. This was followed by SEC-SAXS combined with rigid body modelling on a chimeric U2AF/SF1 trimer containing both human and S. pombe components, revealing that U2AF/SF1 is conformationally flexible in solution and favours a more compact, globular form upon binding a 3′ SS RNA.
In addition to the U2AF/SF1 studies, X-ray structures of the S. pombe and Candida albicans p14/SF3B155 complexes were solved revealing that p14 is more conformationally plastic than previously assumed; p14 displaces SF1 during the splicing cycle, therefore these structures contribute to a unified model of 3′ SS recognition.
Ultimately, this thesis is important foundational work for understanding 3′ SS recognition since it is the first published report of the successful expression and purification of the conserved RNA-binding core of the U2AF dimer and U2AF/SF1 trimer complexes.
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- Graduation date
- Spring 2023
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- Type of Item
- Thesis
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- Degree
- Doctor of Philosophy
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- 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.