The genetic regulatory mechanisms that specify and maintain the differentiated states of cells constitute a central problem in the study of development. Their study is of practical importance for understanding pathological processes that disrupt morphogenesis, cell differentiation and the control of cell proliferation. The long term objective of this project is to understand how homeotic genes specify position-specific cellular fates during development. The immediate goal is to characterize the alternative splicing mechanisms that generate and regulate the structural and functional diversity of proteins encoded by the Ultrabithorax (Ubx) homeotic gene of Drosophila melanogaster. Six UBX protein isoforms are generated by developmentally regulated alternative splicing of the Ubx pre-mRNAs. These isoforms are expressed in complex tissue- and stage- specific patterns that reflect functional specialization of the proteins. Regulated alternative splicing must involve features of the pre-mRNA that define functional differences between splice sites, and trans-acting factors that modulate the relative activities of these sites. Comparisons of exon and intron sequences flanking the splice sites in Ubx pre-mRNAs from different Drosophila species will be used to identify conserved features that may participate in regulation of alternative splicing. In vitro mutagenesis of Ubx minigene constructs will be used to test the importance of these putative cis-acting elements, to characterize their function, and to define additional features of the RNA necessary for proper regulation of splicing. Complementary biochemical and genetic approaches will be used to identify and characterize the trans-acting factors that regulate splicing of the Ubx pre-mRNAs. Affinity adsorption and biochemical fractionation methods, together with in vitro splicing complementation assays, will be used to identify and purify regulatory factors that interact directly with the Ubx pre-mRNAs or with the splicing machinery. These factors will be characterized biochemically and functionally and their developmental distribution will be analyzed. Genetic screens will be performed to identify mutations that act in trans to alter the regulation of Ubx pre-mRNA splicing, thus defining loci that potentially encode splicing factors or their regulators. Subsequent genetic analysis will aim to characterize the function of the identified loci and to facilitate their eventual molecular cloning. These investigations will be important for understanding how Ultrabithorax function is modulated to establish appropriate cellular fates in diverse developmental contexts. Alternative splicing of pre-mRNAs is an important and widespread mechanism for quantitative and qualitative modulation of gene function during development, yet current understanding of splice site selection and its regulation is incomplete. Thus, the proposed investigations will also improve our understanding of an important and widespread genetic regulatory strategy.
