The experiments described in this proposal are designed to gain insight into both the mechanism and regulation of pre-mRNA splicing. Much of the work focuses on a family of conserved proteins known as SR proteins that are both essential for splicing in vitro and also able to modulate the use of alternative splice sites. Studies on the role of snRNAs in splicing catalysis will also be pursued.
Four Specific Aims are proposed. 1. ANALYSIS OF SR PROTEIN FUNCTION IN VITRO. Studies will focus on abundant SR proteins including the prototype ASF/SF2, as well as SC35, SRp20 and SRp40. Several outstanding issues will be addressed. One is the question of specificity, both with respect to protein-RNA and protein-protein interactions. A number of assays will be employed to detect differences in the behavior of individual proteins. The effects of phosphorylation of the RS domain on RNA and protein binding will also be examined. 2. ANALYSIS OF SR PROTEIN FUNCTION IN VIVO. Transfection assays involving overexpression of SR proteins in mammalian cells will be continued to provide further insight into the role of SR proteins in modulating splicing, including autoregulation, and in other recently suggested processes such as nuclear-cytoplasmic transport. Cell lines producing different amounts of ASF/SF2 will produced using the chicken B cell line DT40, and effects on cell growth and splicing will be determined. 3. FUNCTION AND REGULATION OF CLK KINASES. Clk/Sty kinase is the prototype of a family of kinases containing N-terminal RS regions and C-terminal catalytic domains. Recent studies showing that Clk/Sty binds to certain SR proteins, phosphorylates serines in the RS domain, and modulates splice site selection in vitro and in vivo will be pursued. The possibility that different Clk family members target distinct SR proteins will be examined. Regulation of the kinases themselves will be studies. 4. FUNCTION OF SNRNAs IN SPLICING CATALYSIS. Genetic studies involving transfection assays will be continued to investigate further the U2-U6-pre-mRNA interactions required to catalyze splicing. The requirements of mutually exclusive base pairing involving U2, U6 and the pre-mRNA will be pursued, as will studies of an intramolecular U6 helix that may be critical for catalysis. Interactions between purified U2, U6 and a model pre-RNA will be studied in vitro.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM048259-13
Application #
6018926
Study Section
Molecular Biology Study Section (MBY)
Project Start
1992-07-01
Project End
2001-06-30
Budget Start
1999-07-01
Budget End
2000-06-30
Support Year
13
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Columbia University (N.Y.)
Department
Biology
Type
Other Domestic Higher Education
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10027
Li, Huang; Wang, Zhijia; Zhou, Xuexia et al. (2013) Far upstream element-binding protein 1 and RNA secondary structure both mediate second-step splicing repression. Proc Natl Acad Sci U S A 110:E2687-95
Chen, Mo; Manley, James L (2009) Mechanisms of alternative splicing regulation: insights from molecular and genomics approaches. Nat Rev Mol Cell Biol 10:741-54
Feng, Ying; Valley, Matthew T; Lazar, Josef et al. (2009) SRp38 regulates alternative splicing and is required for Ca(2+) handling in the embryonic heart. Dev Cell 16:528-38
Ali, Gul Shad; Palusa, Saiprasad G; Golovkin, Maxim et al. (2007) Regulation of plant developmental processes by a novel splicing factor. PLoS One 2:e471
Li, Xialu; Niu, Tianhui; Manley, James L (2007) The RNA binding protein RNPS1 alleviates ASF/SF2 depletion-induced genomic instability. RNA 13:2108-15
Shi, Yongsheng; Reddy, Bharat; Manley, James L (2006) PP1/PP2A phosphatases are required for the second step of Pre-mRNA splicing and target specific snRNP proteins. Mol Cell 23:819-29
Marin-Vinader, Laura; Shin, Chanseok; Onnekink, Carla et al. (2006) Hsp27 enhances recovery of splicing as well as rephosphorylation of SRp38 after heat shock. Mol Biol Cell 17:886-94
Li, Xialu; Wang, Jin; Manley, James L (2005) Loss of splicing factor ASF/SF2 induces G2 cell cycle arrest and apoptosis, but inhibits internucleosomal DNA fragmentation. Genes Dev 19:2705-14
Li, Xialu; Manley, James L (2005) Inactivation of the SR protein splicing factor ASF/SF2 results in genomic instability. Cell 122:365-78
Millhouse, Scott; Manley, James L (2005) The C-terminal domain of RNA polymerase II functions as a phosphorylation-dependent splicing activator in a heterologous protein. Mol Cell Biol 25:533-44

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