Pre-mRNA splicing must occur with high fidelity to prevent catastrophic errors. Yet, the molecular mechanisms of fidelity in splicing are understood poorly. Splicing is catalyzed by the spliceosome, a dynamic ribonucleoprotein machine in which small nuclear RNA (snRNA) components play key roles in substrate recognition and catalysis. The long-term goals of this proposal are to understand how fidelity is established in splicing and how spliceosome dynamics promote fidelity.
We aim to understand how the spliceosome promotes splicing of a genuine substrate and antagonizes splicing of an aberrant substrate. Further, we aim to understand how splicing of a genuine substrate and stalling of an aberrant substrate each lead to spliceosome disassembly. Recently, we have made significant breakthroughs in understanding how the U2 and U6 snRNAs promote splicing of a genuine substrate, how the DExD/H box ATPase Prp22 antagonizes splicing of an aberrant substrate and how spliceosome disassembly is regulated. Specifically, we aim (i) to determine the role of Prp22 in antagonizing aberrant intermediates, (ii) to determine the roles of the DExD/H box ATPases Prp43 and Brr2 and the EF-2-like GTPase Snu114 in spliceosome disassembly, (iii) to investigate the role of U2/U6 helix I sequences in promoting splicing and (iv) to determine the role of the U2 loop Ha in remodeling the spliceosome for exon ligation. We will pursue these aims using a combined approach of molecular genetics and biochemistry. These studies will likely have broad implications for understanding (i) the function and regulation of DExD/H box ATPases, (ii) the mechanisms for establishing fidelity in splicing and (iii) the mechanisms for regulating the activity of the spliceosome. Further, as fidelity is intimately linked to splice site choice, these studies promise to impact our understanding of the mechanisms for regulating splicing. To investigate splicing, we will utilize the model organism baker's yeast, which resembles a simplified human cell and which has served as a long-standing model for human disease. Significantly, as life requires the faithful expression of genes, splicing errors commonly lead to human disease. Thus, these studies will lead to an understanding of how fidelity is established in human cells, how compromised fidelity may contribute to disease and how fidelity might be engineered to treat disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM062264-08
Application #
7571699
Study Section
Molecular Genetics C Study Section (MGC)
Program Officer
Bender, Michael T
Project Start
2001-03-01
Project End
2011-02-28
Budget Start
2009-03-01
Budget End
2010-02-28
Support Year
8
Fiscal Year
2009
Total Cost
$298,782
Indirect Cost
Name
University of Chicago
Department
Genetics
Type
Schools of Medicine
DUNS #
005421136
City
Chicago
State
IL
Country
United States
Zip Code
60637
He, Yangzi; Staley, Jonathan P; Andersen, Gregers Rom et al. (2017) Structure of the DEAH/RHA ATPase Prp43p bound to RNA implicates a pair of hairpins and motif Va in translocation along RNA. RNA 23:1110-1124
Qin, Daoming; Huang, Lei; Wlodaver, Alissa et al. (2016) Sequencing of lariat termini in S. cerevisiae reveals 5' splice sites, branch points, and novel splicing events. RNA 22:237-53
Soucek, Sharon; Zeng, Yi; Bellur, Deepti L et al. (2016) The Evolutionarily-conserved Polyadenosine RNA Binding Protein, Nab2, Cooperates with Splicing Machinery to Regulate the Fate of pre-mRNA. Mol Cell Biol :
Semlow, Daniel R; Blanco, Mario R; Walter, Nils G et al. (2016) Spliceosomal DEAH-Box ATPases Remodel Pre-mRNA to Activate Alternative Splice Sites. Cell 164:985-98
Wlodaver, Alissa M; Staley, Jonathan P (2014) The DExD/H-box ATPase Prp2p destabilizes and proofreads the catalytic RNA core of the spliceosome. RNA 20:282-94
Koodathingal, Prakash; Staley, Jonathan P (2013) Splicing fidelity: DEAD/H-box ATPases as molecular clocks. RNA Biol 10:1073-9
Kannan, Ram; Hartnett, Sean; Voelker, Rodger B et al. (2013) Intronic sequence elements impede exon ligation and trigger a discard pathway that yields functional telomerase RNA in fission yeast. Genes Dev 27:627-38
Semlow, Daniel R; Staley, Jonathan P (2012) Staying on message: ensuring fidelity in pre-mRNA splicing. Trends Biochem Sci 37:263-73
Nielsen, Klaus H; Staley, Jonathan P (2012) Spliceosome activation: U4 is the path, stem I is the goal, and Prp8 is the keeper. Let's cheer for the ATPase Brr2! Genes Dev 26:2461-7
Mayas, Rabiah M; Maita, Hiroshi; Semlow, Daniel R et al. (2010) Spliceosome discards intermediates via the DEAH box ATPase Prp43p. Proc Natl Acad Sci U S A 107:10020-5

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