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. I propose to study neuronal exon splicing with a focus on the regulatory protein neuronal Polypyrimidine Tract Binding Protein (nPTB). nPTB and its homolog PTB are splicing repressors for multiple exons. The expression of PTB and nPTB are mutually exclusive with PTB restricted to non-neuronal lineages and nPTB found only in post mitotic neurons. During neuronal differentiation PTB is replaced by nPTB. This switch reprograms the splicing of a large set of alternative exons in neurons. The regulation of the neuron specific N1 exon of the c-src has been constructed in vitro. PTB represses the splicing of N1 exon. However neuronal PTB does not repress the splicing of N1 and other neuronal exons. Experiments will examine how this highly homologous protein differs in activity. I will use the N1 model system to analyze how PTB and nPTB differ in their effect on pre-spliceosomal complex assembly. Extracts of cells that have been depleted of PTB and only contain nPTB will be used to analyze for differences in components and conformation compared to the repressed PTB complexes. The determinants of PTB repression will be examined through chimeric PTB / nPTB constructs that will be assayed in vivo and in vitro for repression, binding and complex formation. The Structure of the higher order PTB-RNA and nPTB -RNA complexes will be explored by electron microscopy and X-ray crystallography. Through these experiments I hope to understand in molecular detail how these two highly similar proteins generate different splicing outcomes and thus affect 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.
Misregulation of alternative splicing causes human neurological diseases including Spinal Muscular Atrophy and Frontal Temporal Dementia. For these diseases to be approached therapeutically, an understanding is needed on the mechanisms of splicing regulation and its role in neuronal function.
|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|