Precursor messenger RNA splicing is an essential step in gene expression that can be highly regulated by alternative splicing in higher organisms. While biochemical approaches have been successful in identifying protein factors involved in the regulation of splice site choice in metazoans, there is always the concern that in vitro approaches using extracts from cell lines do not identify the true developmental and tissue-specific regulators of splice site choice. Directed genetic approaches to studying alternative splicing in metazoan model systems, which could reveal the specific regulators, have been few. C. elegans presents us with an exciting opportunity to study splicing and its regulation. The complete genome sequence and over 100,000 cDNA sequences present a unique opportunity for a computational approach to identify and characterize splicing regulatory sequences. The well-developed genetic system could be used to identify genes that regulate specific splicing. The molecular transformation and cytological tools such as the use of green fluorescent protein provide opportunities to engineer reporter genes through which splicing can be monitored with both visual and molecular means. In this proposal we present a genetic, molecular and bioinformatics approach to analyze splicing regulation in C. elegans. The specific aims are to: 1- identify and characterize genes involved in the regulation of cryptic splice site choice. These types of genes can have important consequences in human disease display. 2- use a novel genetic screen, involving visualization of alternative splicing through the use of a green fluorescent protein reporter, to identify trans-acting factors and cis regulatory elements involved in the developmental regulation of alternative splicing of the let-2 gene. 3- use a collection of 845 alternatively spliced genes we have identified computationally in C. elegans to computationally identify cis elements in the pre-mRNA potentially involved in splicing regulation. These putative elements will be characterized using an in vivo splicing reporter method that we have developed.