Pre-mRNA splicing is an almost ubiquitous feature of gene expression. Introns must be properly removed for protein synthesis to take place correctly. Introns are recognized by trans-acting splicing factors during the early steps of spliceosome assembly, and this recognition ensures that pre-mRNA does not escape to the cytoplasm before intron removal. Splice sites are also identified at this early splicing step, and their identification contributes to the fidelity of splicing and the choice of splice site partners, namely, which exons are chosen to be ligated together. Many diseases are caused by difficulties with splice site selection, and regulation of viral gene expression during infection by HIV is dependent upon aspects of this pre-mRNA recognition and transport system. In yeast, mammalian, and viral systems, Ul snRNP is an important factor in these early splice site identification events. The nature of the yeast Ul snRNP-pre-mRNA interaction will be investigated in vitro, and the genetic advantages of yeast will be applied to the study of this snRNP and its constituents. Major foci are the structure and function of several Ul snRNP proteins, including the well studied UIA protein. We are also interested in testing the hypothesis that pre-mRNA structure plays an important role in these early recognition-identification events. pre-mRNA will be structure probed in vivo, and the effects of model structures on splicing assessed. Genetic methods will be applied to identify proteins that might modulate pre-mRNA structure or splice site selection.