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Regulation of Adenovirus Late Region 1 Splicing
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. (Göran Akusjärvi)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. (Göran Akusjärvi)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. (Göran Akusjärvi)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. (Göran Akusjärvi)
(English)Manuscript (Other academic)
Abstract [en]

The major late transcription unit (MLTU) produces one pre-mRNA that is processed into more than 20 cytoplasmic mRNAs by alternative polyadenylation and extensive alternative 3' splice site usage. The alternative splicing of the MLTU is temporally regulated, resulting in the expression of only one mRNA, the L1 52,55K, before the onset of viral genome replication. The L1 unit also encodes the IIIa mRNA, the expression of which is highly regulated at the level of splicing. We have previously shown that the adenoviral L4-33K protein enhances IIIa splicing via the IIIa virus infection-dependent splicing enhancer element. In this study we show that serine to glycine mutations in the tiny RS domain of the L4-33K protein retain more activity in vivo compared to results obtained previously in vitro. In addition, it is also clear that these mutations in the RS domain affect the sub-cellular localization of L4-33K. Thus, the RS domain appears to contain a nuclear localisation signal that is dependent on the serine residues. In a previous report we showed that, in extracts prepared from adenovirus-infected cells, splicing is independent of the general splicing factor U2AF. In this study we also demonstrate that none of the tested U2AF-replacement candidate proteins (PUF60, Caper α, and Caper β) collaborate with L4-33K in the activation of IIIa splicing. It has been suggested that regulation of L1 alternative splicing does not require cis-competition between the 52.55K and IIIa 3' splice sites. We find that activity of the IIIa splice site increases considerably in the absence of cis-competition with the 52,55K splice site. Interestingly, this cis-competition is not virus-specific since this observation is reproducible in a transcription unit where the β-globin 3' splice site replaces the natural 52,55K 3' splice site. We conclude that L1 alternative splicing conforms to the general rule in that it ordinarily makes use of the proximal 3' splice site (52,55K), whereas activation of distal 3' splice site usage requires active intervention. In adenovirus this intervention is achieved by production of the L4-33K protein.

Keyword [en]
Adenovirus, L4-33K, Splicing, 3VDF
Identifiers
URN: urn:nbn:se:uu:diva-101308OAI: oai:DiVA.org:uu-101308DiVA: diva2:212566
Available from: 2009-04-22 Created: 2009-04-22 Last updated: 2011-06-28
In thesis
1. Regulation of Adenoviral Gene Expression by the L4-33K and L4-22K Proteins
Open this publication in new window or tab >>Regulation of Adenoviral Gene Expression by the L4-33K and L4-22K Proteins
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The splicing pattern during an adenovirus infection is shifted at the late phase towards using weaker splice sites, splicing out larger introns. Splicing of weak 3´ splice sites usually requires recognition of the 3´AG dinucleotide before the first catalytic step of splicing. Such splicing events are said to be AG-dependent and requires an interaction of both subunits of the cellular splicing factor U2AF with the 3´ splice site. We show that splicing of transcripts that are AG-dependent in uninfected nuclear extracts (NE) becomes AG-independent in nuclear extracts prepared form adenovirus late-infected HeLa cells (Ad-NE). Further we demonstrate that the first step in splicing of a model transcript, IgM, becomes completely U2AF-independent in Ad-NE. This finding supports our working model that 3´ splice site recognition in Ad-NE is altered, and in fact might be U2AF-independent.

We further show that the adenovirus late protein L4-33K acts as a virus encoded alternative splicing factor. L4-33K activates splicing of both cellular and viral transcripts containing weak 3´ splice sites. This supports the hypothesis that adenovirus alter splicing during the infection to favour usage of weak, suboptimal 3´ splice sites. However, we were unable to find an alternative U2AF-related factor that could stimulate L4-33K splicing enhancer activity. Furthermore, we demonstrate that the serine residues in the C-terminal part of L4-33K are important for the splicing enhancer activity but also for its nuclear localisation.

The adenovirus major late promoter is highly activated after the onset of viral genome replication. Protein complexes binding to downstream elements of the promoter are required for full enhancement of this promoter. We show that an L4-33K-related protein, L4-22K, stimulates transcription from the major late promoter. This stimulation is mainly via the downstream elements and does not require the viral IVa2 protein, which is a transcription factor of the major late promoter.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 45 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 458
National Category
Microbiology in the medical area
Research subject
Medical Virology
Identifiers
urn:nbn:se:uu:diva-101324 (URN)978-91-554-7529-1 (ISBN)
Public defence
2009-06-04, C10:305, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2009-05-13 Created: 2009-04-22 Last updated: 2011-02-24Bibliographically approved

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Backström, EllenorAkusjärvi, Göran

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