Regulation of Adenovirus Alternative Pre-mRNA Splicing

Yue, B-G. 2000. Regulation of adenovirus alternative pre-mRNA splicing. Functional characterization of exonic and intronic splicing enhancer elements. Acta Universitatis Upsaliensis. Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 926. 55pp. Uppsala. ISBN 91-554-4712-0 Pre-mRNA splicing and alternative pre-mRNA splicing are key regulatory steps controlling gene expression in higher eukaryotes. The work in this thesis was focused on a characterization of the significance of exonic and intronic splicing enhancer elements for premRNA splicing. Previous studies have shown that removal of introns with weak and regulated splice sites require a splicing enhancer for activity. Here we extended these studies by demonstrating that two “strong” constitutively active introns, the adenovirus 52,55K and the Drosophila Ftz introns, are absolutely dependent on a downstream splicing enhancer for activity in vitro. Two types splicing enhancers were shown to perform redundant functions as activators of splicing. Thus, SR protein binding to an exonic splicing enhancer element or U1 snRNP binding to a downstream 5 ́splice site independently stimulated upstream intron removal. The data further showed that a 5 ́splice site was more effective and more versatile in activating splicing. Collectively the data suggest that a U1 enhancer is the prototypical enhancer element activating splicing of constitutively active introns. Adenovirus IIIa pre-mRNA splicing is enhanced more than 200-fold in infected extracts. The major enhancer element responsible for this activation was shown to consist of the IIIa branch site/polypyrimidne tract region. It functions as a Janus element and blocks splicing in extracts from uninfected cells while functioning as a splicing enhancer in the context of infected extracts. Phosphorylated SR proteins are essential for pre-mRNA splicing. Large amount recombinant SR proteins are needed in splicing studies. A novel expression system was developed to express phosphorylated, soluble and functionally active ASF/SF2 in E. coli. Bai-Gong Yue, Department of Medical Biochemistry and Microbiology, Biomedical Center, Uppsala University, Box 582, SE-751 23 Uppsala, Sweden