The majority of messenger RNA sequences in eukaryotes are in fragments that need to be correctly spliced in order to be translated into functional proteins. Defects in pre-mRNA splicing have been linked to many human diseases including neurological dysfunction and cancer. The broad, long-term objectives of this grant are to understand the structure and function of the large ribonucleoprotein complex, called the spliceosome, that carries out the pre-mRNA splicing reaction. The spliceosome field has made tremendous progress since its conception almost 20 years ago; however, it is still not clear what factors exist in the core of the spliceosome and how the core is activated or remodeled for catalysis. This proposal will investigate the role of the U6 small nuclear RNA in spliceosome catalysis as well as the function of extrinsic splicing protein factors at the spliceosome core.
Specific aim 1 is to study the 3' intramolecular stem-loop (ISL) of yeast U6 snRNA, which binds a magnesium ion required for the first transesterification reaction. We will test whether the magnesium at the U6/3'ISL is at the 5' splice site of the pre-mRNA by using double sulfur substitutions and rescuing with a thiophilic metal ion. We will use crosslinking strategies to test whether the U6/3'ISL is held in the spliceosome core by RNA or by protein.
Specific aim 2 is to study how an RNA helicase called Prp2 remodels the core of the spliceosome. We will investigate how a prp2-suppressor protein or an intermolecular U2/U6 helix RNA contributes to the spliceosome recognition by Prp2. We will also use crosslinking and yeast genetics approaches to test the interaction between Prp2 and the spliceosomal RNAs.
The specific aim 3 is to investigate whether the activation of the spliceosome by Prp2 is conserved in human. We will inhibit DBP2 by expressing a small interference RNA (siRNA) or a dominant-negative mutant targeting DBP2 in human cells. We will analyze the splicing machinery when DBP2 function is impaired in vivo or in vitro. The proposed study shall reveal the dynamic features and the remodeling of the spliceosome core in yeast and human.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM040639-13
Application #
6738072
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Rhoades, Marcus M
Project Start
1988-08-01
Project End
2007-03-31
Budget Start
2004-04-01
Budget End
2005-03-31
Support Year
13
Fiscal Year
2004
Total Cost
$358,577
Indirect Cost
Name
City of Hope/Beckman Research Institute
Department
Type
DUNS #
027176833
City
Duarte
State
CA
Country
United States
Zip Code
91010
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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