The broad, long-term objectives of this research project are to study RNA metabolism and gene expression. Areas such as RNA processing , RNA transport, and RNA stability and degradation will eventually be explored. Initially, the process of pre-mRNA splicing (i.e., the precise excision of introns from messenger RNA precursors) will be studied. Specifically, the in vitro pre-mRNA splicing system that we developed in the yeast, Saccharomyces cerevisiae, will be utilized to identify and characterize the components in the splicing machinery (i.e., the extrinsic splicing factors and the functional spliceosomes). The results of this study should provide clue to health-related problems which are resulted from abnormal gene expression due to defects in the RNA splicing process. Three extrinsic protein factors required to convert the pre-mRNA on a functional spliceosome to spliced products have been identified by analyzing extracts isolated from several temperature-sensitive rna mutants. Efforts will be made to purify these extrinsic factors by biochemical fractionation of wild-type or overproducer extracts. Antibodies against purified proteins will be raised. The corresponding genes will be isolated by screening yeast DNA library with the antibodies or synthetic oligonucleotides. DNA sequences will be determined, the homology with other genes searched, and the derived protein sequences analyzed. The purified extrinsic factors will be used to examine and to define the functional spliceosomes isolated under different conditions. The roles of ATP in the splicing pathway will be investigated. Attempt will be made to identify splicing factors which may be phosphorylated or adenylated during the conversion of the spliceosome. The interactions among the spliceosome, the extrinsic factors, and the RNA will be investigated by using immunoprecipitation and RNase protection assays. Ultimately, the machinery for nuclear pre-mRNA splicing may be revealed.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM040639-02
Application #
3298398
Study Section
Molecular Biology Study Section (MBY)
Project Start
1988-08-01
Project End
1991-07-31
Budget Start
1989-08-01
Budget End
1990-07-31
Support Year
2
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Texas Austin
Department
Type
Schools of Arts and Sciences
DUNS #
City
Austin
State
TX
Country
United States
Zip Code
78713
Chandra, Manasa; Zang, Shengbing; Li, Haiqing et al. (2012) Nuclear translocation of type I transforming growth factor ýý receptor confers a novel function in RNA processing. Mol Cell Biol 32:2183-95
Gencheva, Marieta; Lin, Ting-Yu; Wu, Xiwei et al. (2010) Nuclear retention of unspliced pre-mRNAs by mutant DHX16/hPRP2, a spliceosomal DEAH-box protein. J Biol Chem 285:35624-32
Gencheva, Marieta; Kato, Mitsuo; Newo, Alain N S et al. (2010) Contribution of DEAH-box protein DHX16 in human pre-mRNA splicing. Biochem J 429:25-32
Dery, Kenneth J; Yean, Shyue-Lee; Lin, Ren-Jang (2008) Assembly and glycerol gradient isolation of yeast spliceosomes containing transcribed or synthetic U6 snRNA. Methods Mol Biol 488:41-63
Huang, Ching-Jung; Tang, Zhaohua; Lin, Ren-Jang et al. (2007) Phosphorylation by SR kinases regulates the binding of PTB-associated splicing factor (PSF) to the pre-mRNA polypyrimidine tract. FEBS Lett 581:223-32
Li, Chunxia; Kato, Mitsuo; Shiue, Lily et al. (2006) Cell type and culture condition-dependent alternative splicing in human breast cancer cells revealed by splicing-sensitive microarrays. Cancer Res 66:1990-9
Silverman, Edward J; Maeda, Ayaka; Wei, Janet et al. (2004) Interaction between a G-patch protein and a spliceosomal DEXD/H-box ATPase that is critical for splicing. Mol Cell Biol 24:10101-10
Edwalds-Gilbert, Gretchen; Kim, Dong-Ho; Silverman, Edward et al. (2004) Definition of a spliceosome interaction domain in yeast Prp2 ATPase. RNA 10:210-20
Silverman, Edward; Edwalds-Gilbert, Gretchen; Lin, Ren-Jang (2003) DExD/H-box proteins and their partners: helping RNA helicases unwind. Gene 312:1-16
Edwalds-Gilbert, G; Kim, D H; Kim, S H et al. (2000) Dominant negative mutants of the yeast splicing factor Prp2 map to a putative cleft region in the helicase domain of DExD/H-box proteins. RNA 6:1106-19

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