Alternative pre-messenger RNA splicing is a critical means of eukaryotic gene regulation that allows a single gene to produce a variety of mRNAs and proteins. Many proteins important for neuronal development and activity are functionally diversified through the differential inclusion of alternative exons. In spite of its importance to neuronal function and disease, the mechanisms controlling alternative splicing are poorly understood. We propose to continue our studies of neuronal exon splicing as mediated by the two regulatory proteins: Polypyrimidine Tract Binding Protein (PTB) and its neuronal homolog nPTB. We have shown that these proteins control large overlapping sets of splicing events during neuronal differentiation, with some exons affected by both proteins and some exons controlled only by PTB. Although most often studied as splicing repressors, PTB and perhaps nPTB can also activate some exons for splicing. We will now examine the mechanistic basis of PTB and nPTB function. We will analyze the differential targeting of exons by PTB and nPTB, and study how they can affect splicing both positively and negatively. Using an in vitro system that reconstructs the regulation of the neuron-specific N1 exon of c-src, we will analyze how PTB blocks spliceosome assembly on its target exons, and identify its important interactions within repressed exon complexes. A particular focus will be a newly identified PTB interaction with the U1 snRNA. Pre-mRNPs, within which splicing regulators function, are very large and complex. We will develop new methods for analyzing these pre-mRNPs both in vitro and in vivo. We will also complete a statistical model of PTB binding and regulation across the genome and extend this analysis to nPTB. Through these experiments, we hope to understand both the mechanisms of PTB and nPTB action, and the role these proteins play in neuronal cell biology.

Public Health Relevance

The understanding of alternative splicing is essential to our understanding of multiple forms of genetic disease. Many human disease mutations alter splicing to produce aberrant mRNAs and proteins. Spinal Muscular Atrophy, Myotonic Dystrophy, and Frontal Temporal Dementia are neurologic disorders of splicing regulation. For these diseases to be approached therapeutically, much more information is needed on the mechanisms of splicing regulation and its role in neuronal function.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01GM049662-21
Application #
8691853
Study Section
Molecular Genetics C Study Section (MGC)
Program Officer
Bender, Michael T
Project Start
Project End
Budget Start
Budget End
Support Year
21
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
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Li, Qin; Zheng, Sika; Han, Areum et al. (2014) The splicing regulator PTBP2 controls a program of embryonic splicing required for neuronal maturation. Elife 3:e01201
Pandya-Jones, Amy; Bhatt, Dev M; Lin, Chia-Ho et al. (2013) Splicing kinetics and transcript release from the chromatin compartment limit the rate of Lipid A-induced gene expression. RNA 19:811-27
Zheng, Sika; Damoiseaux, Robert; Chen, Liang et al. (2013) A broadly applicable high-throughput screening strategy identifies new regulators of Dlg4 (Psd-95) alternative splicing. Genome Res 23:998-1007
Zheng, Sika; Gray, Erin E; Chawla, Geetanjali et al. (2012) PSD-95 is post-transcriptionally repressed during early neural development by PTBP1 and PTBP2. Nat Neurosci 15:381-8, S1
Gehman, Lauren T; Meera, Pratap; Stoilov, Peter et al. (2012) The splicing regulator Rbfox2 is required for both cerebellar development and mature motor function. Genes Dev 26:445-60
Tang, Zhen Zhi; Sharma, Shalini; Zheng, Sika et al. (2011) Regulation of the mutually exclusive exons 8a and 8 in the CaV1.2 calcium channel transcript by polypyrimidine tract-binding protein. J Biol Chem 286:10007-16
Sharma, Shalini; Maris, Christophe; Allain, Frederic H-T et al. (2011) U1 snRNA directly interacts with polypyrimidine tract-binding protein during splicing repression. Mol Cell 41:579-88
Pandya-Jones, Amy (2011) Pre-mRNA splicing during transcription in the mammalian system. Wiley Interdiscip Rev RNA 2:700-17
Gehman, Lauren T; Stoilov, Peter; Maguire, Jamie et al. (2011) The splicing regulator Rbfox1 (A2BP1) controls neuronal excitation in the mammalian brain. Nat Genet 43:706-11

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