We review here results arising from the systematic functional analysis of Nova, a neuron-specific RNA binding protein targeted in an autoimmune neurological disorder associated with cancer. We have developed a combination of biochemical, genetic and bioinformatic methods to generate a global understanding of Nova's role as a splicing regulator. Genome-wide identification and validation of Nova target RNAs has yielded unexpected insights into the protein's mechanism of action and into the functionally coherent role of Nova in the biology of the neuronal synapse. These studies provide us with a platform for understanding the role of RNA binding proteins in tissue-specific splicing regulation and in disease.