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.
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.
|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|
|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|
|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|
|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|
|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|
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