Alternative splicing is an important means of genetic control in eucaryotic cells. By altering the splicing pattern of the primary transcript, a variety of proteins can be created from a single gene. Alternative splicing is especially prominent in the mammalian nervous system, where it regulates the production of many proteins that are important for neuronal development, function, and disease. Unfortunately, although our understanding of the general biochemistry of splicing has advanced significantly, much less is known of how splicing is regulated. The mouse c-src gene has provided an effective model system for studying a neuron-specific splicing event. Neurons produce a different form of the src protein from other tissues, resulting from the neuron-specific insertion of an extra exon (the N1 exon) into the src mRNA. Two major cis-acting elements that control N1 splicing have been identified by site specific mutagenesis of a transfected src mini-gene. These are an intronic splicing enhancer, downstream of N1, that activates splicing of the exon but is only partially neural specific, and the 3' splice site upstream of the exon that represses the splicing in non-neuronal cells. The combination of these two sequences confers neural specific splicing on a heterologous test exon. The regulated splicing of the N1 exon was reconstituted in extracts of neuronal and non-neuronal cells and some of the regulatory proteins have been identified. These include the KSRP, hnRNP F, hnRNP H and PTB proteins binding to the downstream enhancer, and the PTB protein also binding to the upstream 3' splice site. How the RNA/protein complexes at these sites combine to generate the precise tissue specific inclusion of an exon is still unclear. This project will pursue the molecular analysis of src neuron-specific splicing. Using a variety of biochemical assays, the assembly of the regulatory proteins onto the enhancer and repressor RNA sequences will be studied, and the interactions of these RNA/protein complexes with each other, and with components of the spliceosome will be dissected. New regulatory proteins will be characterized, including a neural specific form of PTB. The role of the recently described splicing enhancer protein, KSRP, will be studied and the functional domains of the protein delineated. Finally, simplified splicing systems will be developed for the analysis of individual regulatory proteins. Our goal is to understand in molecular detail how a simple change in splicing pattern is regulated in differentiated cells.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular Biology Study Section (MBY)
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Rhoades, Marcus M
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University of California Los Angeles
Schools of Medicine
Los Angeles
United States
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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:
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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
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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
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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