Pre-mRNA splicing is essential for gene expression in all eukaryotes and errors in splicing cause genetic disorders and many other diseases. Mutations in splicing factor Prp8, for example, cause a severe form of human genetic disorder Retinitis Pigmentosa. A thorough understanding of the molecular mechanisms of pre-mRNA splicing has the potential to provide useful approaches for human disease therapy. Splicing of introns is carried out through two transesterification reactions catalyzed by the spliceosome, a large RNA/protein complex composed of five snRNAs and over 100 protein factors. Many lines of evidence point to Prp8 as a key spliceosomal protein that interacts intimately with RNA at the catalytic core, potentially helping the formation and stabilization of the catalytic core. Prp8 is one of the largest and most conserved nuclear proteins known, but it does not have obvious sequence homology with any other known protein. Further structural and biochemical analyses would provide valuable insight into Prp8's function in splicing. However, these studies are hindered by difficulties in obtaining large quantities of full-length Prp8. Identifying, expressing, and purifying domains of Prp8 will provide a valuable alternative approach for characterizing Prp8. This proposal uses a unique high throughput approach to identify domains of Prp8 that can be expressed in soluble forms in E. coli and determine structures of these domains. Structures of these domains and comparison with other known structures can provide important information on the function of Prp8 in splicing, directing future mutational/genetic experiments. These soluble domains are also valuable resources for characterizing Prp8's biochemical properties, such as its interaction with RNA, other protein partners, and among different Prp8 domains. Once structural and biochemical characterizations of individual domains are completed, similar characterizations of regions of Prp8 composing multiple domains can be performed. This approach is a critical step toward generating a complete picture of Prp8 that cannot be obtained otherwise, significantly advancing our understanding of the molecular mechanisms of pre-mRNA splicing. Pre-mRNA splicing is essential for gene expression in all eukaryotes and errors in splicing cause genetic disorders and many other diseases. Mutations in splicing factor Prp8, for example, cause a severe form of human genetic disorder Retinitis Pigmentosa. A thorough understanding of the molecular mechanisms of pre-mRNA splicing has the potential to provide useful approaches for human disease therapy.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM080334-05
Application #
8323472
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Bender, Michael T
Project Start
2008-09-01
Project End
2014-08-31
Budget Start
2012-09-01
Budget End
2014-08-31
Support Year
5
Fiscal Year
2012
Total Cost
$269,920
Indirect Cost
$93,502
Name
University of Colorado Denver
Department
Biochemistry
Type
Schools of Medicine
DUNS #
041096314
City
Aurora
State
CO
Country
United States
Zip Code
80045
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