The central nervous system comprises the tissues and cells with the highest rate of alternative splicing in the body, and RNA-binding proteins play a major functional role in neurons. To better understand the contribution of RNA processing to nerve cell biology, and to help elucidate the function that RNA processing regulators play in neuron physiology and neurologic disorders it is necessary to identify which RNA-binding proteins are involved in these biological pathways, and to characterize how they work at the molecular level. Our long-term goal is to understand the molecular mechanisms regulating protein-RNA networks that control alternative splicing, and how they relate to neuron biology, and to disease of the nervous system. The objective of this proposal is to study the molecular basis of how NOVA, a neuron-specific splicing factor involved in an autoimmune motor disease, regulates nerve cell-specific alternative splicing of the ubiquitous protein agrin - a molecule that is the master architect of nerve-muscle synapses at the neuromuscular junction and that is involved in congenital myasthenic syndrome (CMS) in humans. Our preliminary data indicate that mice that are null for the two Nova1 and Nova2 genes fail to make a nerve-derived splice isoform of agrin - termed Z+ agrin - that is critical for the formation, development, and maintenance of the neuromuscular junction. However, the specific mechanism by which Nova regulates this essential developmental switch is still unknown. The central hypothesis of this proposal is that NOVA directly regulates alternative splicing of agrin at the Z site to shape neuromuscular synapses, and that this splicing switch constitutes a novel entry point for therapeutic intervention in specific disorders of the nervous system. In Aim 1 we will test the hypothesis that a novel intronic splicing enhancer mediates Nova-dependent inclusion of agrin Z exons directly. To tackle this question we have developed a cell-based splicing assay that will allow us to test the function of Nova proteins in combination with agrin minigenes. In Aim 2 we will analyze the consequences of Nova deficiency in the formation of ribonucleoprotein complexes and its relationship to the etiology of nervous system pathologies by using an inducible neuronal cell model system of Nova deficiency. This system addresses the inherent technical difficulties in generating splicing-active extracts from mouse brain, while at the same time it provides a flexible platform to test whether modulation of agrin splicing at the Z site is feasible entry point for therapeutic intervention. Understanding how the Nova-agrin regulatory switch is regulated may have clinical implications in RNA-mediated neurodegenerative disorders, CMS pathology, Alzheimer's disease, and epilepsy. Furthermore, this project will provide both undergraduate and graduate students with a unique opportunity to learn the fundamentals of molecular biology and biomedical research, and help them in their pursue of a career in the biomedical field.