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 with a focus on four regulatory proteins. Polypyrimidine Tract Binding Protein (PTB) and its neuronal homolog nPTB are splicing repressers for multiple exons. The Fox-1 and Fox-2 proteins are enhancers of a different but overlapping set of exons. Previously, the regulation of the neuron-specific N1 exon of c-src was reconstructed in vitro. We will use this system to analyze how PTB represses spliceosome assembly, and identify its target interactions. Neuronal PTB does not repress the splicing of N1 and other neuronal exons. Experiments will examine how this highly homologous protein differs in activity. The Fox proteins activate N1 and other neuronal exons through an important splicing enhancer element UGCAUG. The mechanism of this splicing stimulation will be examined using both in vitro and in vivo approaches. The biology of these regulators in differentiating neurons will be explored through their depletion or mis-expression in cells. Groups of exons controlled by these factors will be identified in microarray experiments and examined for common features and function in common regulatory pathways. The complex posttranscriptional regulation of nPTB by PTB in neurons and glia will also be a focus. Through these experiments, we hope to understand both the mechanisms of these proteins' action, and the role they play in neuronal cell biology. The understanding of alternative splicing is essential to our understanding of multiple forms of genetic disease. Spinal Muscular Atrophy, Myotonic Dystrophy, and Prefrontal Dementia are neurologic disorders of splicing regulation. Many human disease mutations alter splicing regulatory elements to produce aberrant proteins. 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)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM049662-16S1
Application #
7883069
Study Section
Molecular Genetics C Study Section (MGC)
Program Officer
Bender, Michael T
Project Start
2009-08-13
Project End
2011-06-30
Budget Start
2009-08-13
Budget End
2011-06-30
Support Year
16
Fiscal Year
2009
Total Cost
$112,001
Indirect Cost
Name
University of California Los Angeles
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Yeom, Kyu-Hyeon; Mitchell, Simon; Linares, Anthony J et al. (2018) Polypyrimidine tract-binding protein blocks miRNA-124 biogenesis to enforce its neuronal-specific expression in the mouse. Proc Natl Acad Sci U S A 115:E11061-E11070
Ke, Shengdong; Pandya-Jones, Amy; Saito, Yuhki et al. (2017) m6A mRNA modifications are deposited in nascent pre-mRNA and are not required for splicing but do specify cytoplasmic turnover. Genes Dev 31:990-1006
Vuong, John K; Lin, Chia-Ho; Zhang, Min et al. (2016) PTBP1 and PTBP2 Serve Both Specific and Redundant Functions in Neuronal Pre-mRNA Splicing. Cell Rep 17:2766-2775
Wongpalee, Somsakul Pop; Vashisht, Ajay; Sharma, Shalini et al. (2016) Large-scale remodeling of a repressed exon ribonucleoprotein to an exon definition complex active for splicing. Elife 5:
Keppetipola, Niroshika M; Yeom, Kyu-Hyeon; Hernandez, Adrian L et al. (2016) Multiple determinants of splicing repression activity in the polypyrimidine tract binding proteins, PTBP1 and PTBP2. RNA 22:1172-80
Damianov, Andrey; Ying, Yi; Lin, Chia-Ho et al. (2016) Rbfox Proteins Regulate Splicing as Part of a Large Multiprotein Complex LASR. Cell 165:606-19
Zhang, Xiaochang; Chen, Ming Hui; Wu, Xuebing et al. (2016) Cell-Type-Specific Alternative Splicing Governs Cell Fate in the Developing Cerebral Cortex. Cell 166:1147-1162.e15
Vuong, Celine K; Black, Douglas L; Zheng, Sika (2016) The neurogenetics of alternative splicing. Nat Rev Neurosci 17:265-81
Linares, Anthony J; Lin, Chia-Ho; Damianov, Andrey et al. (2015) The splicing regulator PTBP1 controls the activity of the transcription factor Pbx1 during neuronal differentiation. Elife 4:e09268
Sharma, Shalini; Wongpalee, Somsakul Pop; Vashisht, Ajay et al. (2014) Stem-loop 4 of U1 snRNA is essential for splicing and interacts with the U2 snRNP-specific SF3A1 protein during spliceosome assembly. Genes Dev 28:2518-31

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