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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM080334-02
Application #
7678577
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Bender, Michael T
Project Start
2008-09-01
Project End
2013-08-31
Budget Start
2009-09-01
Budget End
2010-08-31
Support Year
2
Fiscal Year
2009
Total Cost
$276,150
Indirect Cost
Name
University of Colorado Denver
Department
Biochemistry
Type
Schools of Medicine
DUNS #
041096314
City
Aurora
State
CO
Country
United States
Zip Code
80045
Zhang, Lingdi; Li, Xueni; Hill, Ryan C et al. (2015) Brr2 plays a role in spliceosomal activation in addition to U4/U6 unwinding. Nucleic Acids Res 43:3286-97
Garrey, Stephen M; Katolik, Adam; Prekeris, Mantas et al. (2014) A homolog of lariat-debranching enzyme modulates turnover of branched RNA. RNA 20:1337-48
Zhang, Lingdi; Li, Xueni; Zhao, Rui (2013) Structural analyses of the pre-mRNA splicing machinery. Protein Sci 22:677-92
Li, Xueni; Zhang, Wenzheng; Xu, Tao et al. (2013) Comprehensive in vivo RNA-binding site analyses reveal a role of Prp8 in spliceosomal assembly. Nucleic Acids Res 41:3805-18
Guarnieri, Michael T; Blagg, Brian S J; Zhao, Rui (2011) A high-throughput TNP-ATP displacement assay for screening inhibitors of ATP-binding in bacterial histidine kinases. Assay Drug Dev Technol 9:174-83
Pazy, Y; Motaleb, M A; Guarnieri, M T et al. (2010) Identical phosphatase mechanisms achieved through distinct modes of binding phosphoprotein substrate. Proc Natl Acad Sci U S A 107:1924-9
Liu, Weizhi; Zhao, Rui; McFarland, Craig et al. (2009) Structural insights into parasite eIF4E binding specificity for m7G and m2,2,7G mRNA caps. J Biol Chem 284:31336-49
Zhang, Lingdi; Xu, Tao; Maeder, Corina et al. (2009) Structural evidence for consecutive Hel308-like modules in the spliceosomal ATPase Brr2. Nat Struct Mol Biol 16:731-9