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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
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab. (Göran Akusjärvi)ORCID iD: 0000-0002-0797-8007
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
2012 (English)In: Virology, ISSN 0042-6822, E-ISSN 1096-0341, Vol. 433, no 2, 273-281 p.Article 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.

Place, publisher, year, edition, pages
2012. Vol. 433, no 2, 273-281 p.
Keyword [en]
L4-33K, L4-22K, splicing, localization, adenovirus, replication centers, transcription sites, peripheral replicative zone
National Category
Microbiology in the medical area
Identifiers
URN: urn:nbn:se:uu:diva-159614DOI: 10.1016/j.virol.2012.08.021ISI: 000310095700001PubMedID: 22944109OAI: oai:DiVA.org:uu-159614DiVA: diva2:445920
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
2. Functional Characterization of the Evolutionarily Conserved Adenoviral Proteins L4-22K and L4-33K
Open this publication in new window or tab >>Functional Characterization of the Evolutionarily Conserved Adenoviral Proteins L4-22K and L4-33K
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Regulation of adenoviral gene expression is a complex process directed by viral proteins controlling a multitude of different activities at distinct phases of the virus life cycle. This thesis discusses adenoviral regulation of transcription and splicing by two proteins expressed at the late phase: L4-22K and L4-33K. These are closely related with a common N-terminus but unique C-terminal domains. The L4-33K protein is an alternative RNA splicing factor inducing L1-IIIa mRNA splicing, while L4-22K is stimulating transcription from the major late promoter (MLP). The L4-33K protein contains a tiny RS-repeat in its unique C-terminal end that is essential for the splicing enhancer function of the protein. Here we demonstrate that the tiny RS-repeat is required for localization of the protein to the nucleus and viral replication centers. Further, we describe an auto-regulatory loop where L4-33K enhances splicing of its own intron. The preliminary characterization of the responsive RNA-element suggests that it differs from the previously defined L4-33K-responsive element activating L1-IIIa mRNA splicing.

L4-22K lacks the ability to enhance L1-IIIa splicing in vivo, and here we show that the protein is defective in L1-IIIa or other late pre-mRNA splicing reactions in vitro. Interestingly, we found a novel function for the L4-22K and L4-33K proteins as regulators of E1A alternative splicing. Both proteins selectively upregulated E1A-10S mRNA accumulation in transfection experiments, by a mechanism independent of the tiny RS-repeat.

Although L4-22K is reported to be an MLP transcriptional enhancer protein, here we show that L4-22K also functions as a repressor of MLP transcription. This novel activity depends on the integrity of the major late first leader 5’ splice site. The model suggests that at low concentrations L4-22K activates MLP transcription while at high concentrations L4-22K represses transcription.

So far, characterizations of the L4-22K and L4-33K proteins have been limited to human adenoviruses 2 or 5 (HAdV-2/5). We expanded our experiments to include HAdV-3, HAdV-4, HAdV-9, HAdV-11 and HAdV-41. The results demonstrated that the transcription- or splicing-enhancing properties of L4-22K and L4-33K, respectively, are evolutionarily conserved and non-overlapping. Thus, the sequence-based conservation is mirrored by the functions, as expected for functionally important proteins.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 74 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1062
Keyword
L4-22K, L4-33K, RNA, splicing, adenovirus, nuclear localization, replication, transcription, evolution, SR protein, MLP, promoter, E1A, serotypes
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:uu:diva-238487 (URN)978-91-554-9132-1 (ISBN)
Public defence
2015-02-13, C8:301, BMC, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2015-01-23 Created: 2014-12-12 Last updated: 2015-03-09

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Östberg, SaraTörmänen Persson, HeidiAkusjärvi, Göran

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