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Two Cellular Protein Kinases, DNA-PK and PKA, Phosphorylate the Adenoviral L4-33K Protein and Have Opposite Effects on L1 Alternative RNA Splicing
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Department of Nutrition, University of Oslo.
Department of Nutrition, University of Oslo.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
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2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, e31871- p.Article in journal (Refereed) Published
Abstract [en]

Accumulation of the complex set of alternatively processed mRNA from the adenovirus major late transcription unit (MLTU) is subjected to a temporal regulation involving both changes in poly (A) site choice and alternative 3´ splice site usage. We have previously shown that the adenovirus L4-33K protein functions as an alternative splicing factor involved in activating the shift from L1-52,55K to L1-IIIa mRNA. Here we show that L4-33K specifically associates with the catalytic subunit of the DNA-dependent protein kinase (DNA-PK) in uninfected and adenovirus-infected nuclear extracts. Further, we show that L4-33K is highly phosphorylated by DNA-PK in vitro in a double stranded DNA-independent manner. Importantly, DNA-PK deficient cells show an enhanced production of the L1-IIIa mRNA suggesting an inhibitory role of DNA-PK on the temporal switch in L1 alternative RNA splicing. Moreover, we show that L4-33K also is phosphorylated by protein kinase A (PKA), and that PKA has an enhancer effect on L4-33K-stimulated L1-IIIa splicing. Hence, we demonstrate that these kinases have opposite effects on L4-33K function; DNA-PK as an inhibitor and PKA as an activator of L1-IIIa mRNA splicing. Taken together, this is the first report identifying protein kinases that phosphorylate L4-33K and to suggest novel regulatory roles for DNA-PK and PKA in adenovirus alternative RNA splicing.

Place, publisher, year, edition, pages
2012. e31871- p.
Keyword [en]
L4-33K, adenovirus, splicing, phosphorylation, DNA-PK, DNA-dependent protein kinase, PKA, cAMP-dependent protein kinase, MLTU, L4-22K
National Category
Microbiology in the medical area
Identifiers
URN: urn:nbn:se:uu:diva-159617DOI: 10.1371/journal.pone.0031871ISI: 000302873700113OAI: oai:DiVA.org:uu-159617DiVA: diva2:445914
Available from: 2011-10-05 Created: 2011-10-05 Last updated: 2017-12-08Bibliographically approved
In thesis
1. The Adenovirus L4-33K Protein: A Key Regulator of Virus-specific Alternative Splicing
Open this publication in new window or tab >>The Adenovirus L4-33K Protein: A Key Regulator of Virus-specific Alternative Splicing
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Adenoviruses have been extensively studied in the field of gene regulation, since their genes are subjected to a tightly controlled temporal expression during the virus lifetime. The early-to-late shift in adenoviral gene expression distinguishes two completely different programs in gene expression. The adenoviral L4-33K protein, which is the subject of this thesis, was previously implicated to be a key player in the transition from the early to the late phase of infection. Here we show that L4-33K activates late gene expression by functioning as a virus-encoded alternative RNA splicing factor activating splicing of transcripts containing weak 3’ splice sites; a feature common to the viral genes expressed at late times of infection.

The splicing enhancer activity of L4-33K was mapped to a tiny arginine/serine (RS) repeat in the carboxyl-terminal domain of the protein. Also, the subcellular distribution to the nucleus with enrichment in the nuclear membrane and subnuclear redistribution to viral replication centers during a lytic infection was observed to depend on this motif. RS repeats are common features for the cellular splicing factors serine/arginine-rich (SR) proteins, which in turn are regulated by reversible phosphorylation.

We further show that L4-33K is phosphorylated by two cellular protein kinases, the double-stranded DNA-dependent protein kinase (DNA-PK) and protein kinase A (PKA) in vitro. Interestingly, DNA-PK and PKA have opposite effects on the control of the temporally regulated L1 alternative RNA splicing. DNA-PK functions as an inhibitor of the late specific L1-IIIa pre-mRNA splicing whereas PKA functions as an activator of L1-IIIa pre-mRNA splicing.

In summary, this thesis describes L4-33K as an SR protein related viral alternative splicing factor. A tiny RS repeat conveys splicing enhancer activity as well as redistribution of L4-33K to replication centers. Finally, DNA-PK and PKA that phosphorylates L4-33K are suggested to be novel regulatory factors controlling adenovirus alternative splicing.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 66 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 709
Keyword
L4-33K, adenovirus, splicing, phosphorylation, localization, replication centers, L4-22K, MLTU, DNA-PK, DNA-dependent protein kinase, PKA, cAMP-dependent protein kinase, transcription sites, SR protein
National Category
Microbiology in the medical area
Research subject
Medical Virology
Identifiers
urn:nbn:se:uu:diva-159632 (URN)978-91-554-8178-0 (ISBN)
Public defence
2011-11-17, C10:301, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2011-10-26 Created: 2011-10-05 Last updated: 2011-11-04Bibliographically approved

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Törmänen Persson, HeidiPunga, TanelEngström, ÅkeAkusjärvi, Göran

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