Alternative splicing of precursor-RNA is a common genetic regulatory mechanism that gives rise to multiple protein isoforms with distinct functional properties. Alternative splicing is particularly common in excitable cells such as nerve and muscle where many of the genes encoding ion channels and muscle contractile proteins show extremely complex tissue-and cell-type specific splicing patterns. These gene products play critical roles in determining properties of excitability and contractility suggesting that alternative splicing is a key regulatory step in the expression of genes essential for maintenance of normal function of nerve and muscle. Most studies of alternative splicing rely on in vitro systems. As a result, little is know about the cis-elements or trans-acting factors that act in concert to determine tissue-specific splicing patterns. In the research described here, the Shaker (Sh) gene of Drosophila melanogaster is being used as a model system to examine the in vivo mechanisms of regulation of alternative splicing that result in tissue-specific splicing that result in tissue-specific expression of kinetically distinct potassium ion (K+) channels. Using lacZ as a reporter gene for regulated splicing activity in transgenic animals, we have demonstrated that a 240 nuc. intron region contains the cis-elements required for correct splice choice in the indirect flight muscles and that splicing in this tissue is positively regulated. The cis-elements will be more precisely defined by in vivo analysis of splicing patterns in transformants carrying deletions and insertions of this 240 nuc. region and by sequence comparison to the corresponding introns from Drosophila virilis. Once the cis-elements have been defined, the project will be extended to identify the trans-acting factors that recognize and bind to the cis-ehancer by UV crosslinking and Northwestern analysis of nuclear proteins to synthetic RNA containing the cis-elements. Isolation of cDNA clones encoding the trans-acting splicing factors that recognize and bind to the cis-will be accomplished either by direct screening of an embryonic expression library using the cis-elements as probes, screening an expression library using a yeast three-hybrid system, or biochemical purification followed by peptide sequence determination and screening the library by conventional hybridization methods. The results of these experiments will provide a crucial link between in vitro and in vivo splicing studies and are expected to be applicable to mechanisms of alternative splicing in all organisms. The importance of examining the regulation of alternative splicing of a K+ channel gene is underscored by recent evidence indicating that many of the mammalian K+ channels genes are alternatively spliced and a number of human hereditary diseases of muscle including Long QT syndrome.
