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The Adenovirus L4-33K Protein: A Key Regulator of Virus-specific Alternative Splicing
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. (Göran Akusjärvi)
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. , p. 66
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 709
Keyword [en]
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: urn:nbn:se:uu:diva-159632ISBN: 978-91-554-8178-0 (print)OAI: oai:DiVA.org:uu-159632DiVA, id: diva2:445961
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: 2018-01-12Bibliographically approved
List of papers
1. L4-33K, an adenovirus-encoded alternative RNA splicing factor
Open this publication in new window or tab >>L4-33K, an adenovirus-encoded alternative RNA splicing factor
2006 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, no 48, p. 36510-36517Article in journal (Refereed) Published
Abstract [en]

Splicing of the adenovirus IIIa mRNA is subjected to a strict temporal regulation during virus infection such that efficient IIIa 3' splice site usage is confined to the late phase of the infectious cycle. Here we show that the adenovirus L4-33K protein functions as a virus-encoded RNA splicing factor that preferentially activates splicing of transcripts with a weak 3' splice site sequence context, a sequence configuration that is shared by many of the late adenovirus 3' splice sites. Furthermore, we show that L4-33K activates IIIa splicing through the IIIa virus infection-dependent splicing enhancer element (3VDE). This element was previously shown to be the minimal element, both necessary and sufficient, for activation of IIIa splicing in the context of an adenovirus-infected cell. L4-33K stimulates an early step in spliceosome assembly and appears to be the only viral protein necessary to convert a nuclear extract prepared from uninfected HeLa cells to an extract with splicing properties very similar to a nuclear extract prepared from adenovirus late-infected cells. Collectively, our results suggest that L4-33K is the key viral protein required to activate the early to late switch in adenovirus major late L1 alternative splicing.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-22559 (URN)10.1074/jbc.M607601200 (DOI)000242220800006 ()17028184 (PubMedID)
Available from: 2007-01-18 Created: 2007-01-18 Last updated: 2017-12-07Bibliographically approved
2. Serine 192 in the tiny RS repeat of the adenoviral L4-33K splicing enhancer protein is essential for function and reorganization of the protein to the periphery of viral replication centers
Open this publication in new window or tab >>Serine 192 in the tiny RS repeat of the adenoviral L4-33K splicing enhancer protein is essential for function and reorganization of the protein to the periphery of viral replication centers
2012 (English)In: Virology, ISSN 0042-6822, E-ISSN 1096-0341, Vol. 433, no 2, p. 273-281Article in journal (Refereed) Published
Abstract [en]

The adenovirus L4-33K protein is a key regulator involved in the temporal shift from early to late pattern of mRNA expression from the adenovirus major late transcription unit. L4-33K is a virus-encoded alternative splicing factor, which enhances processing of 3’ splice sites with a weak sequence context. Here we show that L4-33K expressed from a transfected plasmid has a diffuse nuclear localization with strong enrichment in the nuclear membrane. We further show that the highly conserved part of the carboxy-terminal end of L4-33K, which functions as the splicing enhancer domain, is sufficient for nuclear localization of the protein. Interestingly, infection of the transfected cells caused a redistribution of L4-33K from the nuclear membrane into discrete ring-like structures corresponding to the viral transcription sites. We also show that serine 192 in the tiny RS repeat, which is critical for the splicing enhancer function of L4-33K, is necessary for the nuclear localization and redistribution of L4-33K protein into viral transcription sites. Collectively, our results show a good correlation between the activity of L4-33K as a splicing enhancer protein and its localization to viral transcription sites.

Keyword
L4-33K, L4-22K, splicing, localization, adenovirus, replication centers, transcription sites, peripheral replicative zone
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:uu:diva-159614 (URN)10.1016/j.virol.2012.08.021 (DOI)000310095700001 ()22944109 (PubMedID)
Available from: 2011-10-05 Created: 2011-10-05 Last updated: 2018-01-12Bibliographically approved
3. Two Cellular Protein Kinases, DNA-PK and PKA, Phosphorylate the Adenoviral L4-33K Protein and Have Opposite Effects on L1 Alternative RNA Splicing
Open this publication in new window or tab >>Two Cellular Protein Kinases, DNA-PK and PKA, Phosphorylate the Adenoviral L4-33K Protein and Have Opposite Effects on L1 Alternative RNA Splicing
Show others...
2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, p. e31871-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.

Keyword
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:nbn:se:uu:diva-159617 (URN)10.1371/journal.pone.0031871 (DOI)000302873700113 ()
Available from: 2011-10-05 Created: 2011-10-05 Last updated: 2018-01-12Bibliographically approved

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