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Regulation of adenovirus alternative pre-mRNA splicing: Functional characterization of exonic and intronic splicing enhancer elements
Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
2000 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Pre-mRNA splicing and alternative pre-mRNA splicing are key regulatory steps controlling geneexpression in higher eukaryotes. The work in this thesis was focused on a characterization of thesignificance of exonic and intronic splicing enhancer elements for pre-mRNA splicing.

Previous studies have shown that removal of introns with weak and regulated splice sitesrequire a splicing enhancer for activity. Here we extended these studies by demonstrating thattwo "strong" constitutively active introns, the adenovirus 52,55K and the Drosophila Ftzintrons, are absolutely dependent on a downstream splicing enhancer for activity in vitro.

Two types splicing enhancers were shown to perform redundant functions as activators ofSplicing. Thus, SR protein binding to an exonic splicing enhancer element or U1 snRNP bindingto a downstream 5'splice site independently stimulated upstream intron removal. The datafurther 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 activatingsplicing of constitutively active introns.

Adenovirus IIIa pre-mRNA splicing is enhanced more than 200-fold in infected extracts. Themajor enhancer element responsible for this activation was shown to consist of the IIIa branchsite/polypyrimidne tract region. It functions as a Janus element and blocks splicing in extractsfrom 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 recombinantSR proteins are needed in splicing studies. A novel expression system was developed to expressphosphorylated, soluble and functionally active ASF/SF2 in E. Coli.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2000. , p. 55
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 0282-7476 ; 926
Keywords [en]
Biochemistry, adenovirus, pre-mRNA splicing, splicing enhancer, MLTU, L1, SR protein, ASF/SF2, E. coli, phosphorylation, dephosphorylation
Keywords [sv]
Biokemi
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Microbiology
Identifiers
URN: urn:nbn:se:uu:diva-454ISBN: 91-554-4712-0 (print)OAI: oai:DiVA.org:uu-454DiVA, id: diva2:165076
Public defence
2000-09-22, Sal C10-301, BMC, Uppsala, 13:15
Available from: 2000-09-01 Created: 2000-09-01Bibliographically approved

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