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Cellular splicing and transcription regulatory protein p32 represses adenovirus major late transcription and causes hyperphosphorylation of RNA polymerase II
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
2006 (English)In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 80, no 10, 5010-5020 p.Article in journal (Refereed) Published
Abstract [en]

The cellular protein p32 is a multifunctional protein, which has been shown to interact with a large number of cellular and viral proteins and to regulate several important activities like transcription and RNA splicing. We have previously shown that p32 regulates RNA splicing by binding and inhibiting the essential SR protein ASF/SF2. To determine whether p32 also functions as a regulator of splicing in virus-infected cells, we constructed a recombinant adenovirus expressing p32 under the transcriptional control of an inducible promoter. Much to our surprise the results showed that p32 overexpression effectively blocked mRNA and protein expression from the adenovirus major late transcription unit (MLTU). Interestingly, the p32-mediated inhibition of MLTU transcription was accompanied by an approximately 4.5-fold increase in Ser 5 phosphorylation and an approximately 2-fold increase in Ser 2 phosphorylation of the carboxy-terminal domain (CTD). Further, in p32-overexpressing cells the efficiency of RNA polymerase elongation was reduced approximately twofold, resulting in a decrease in the number of polymerase molecules that reached the end of the major late L1 transcription unit. We further show that p32 stimulates CTD phosphorylation in vitro. The inhibitory effect of p32 on MLTU transcription appears to require the CAAT box element in the major late promoter, suggesting that p32 may become tethered to the MLTU via an interaction with the CAAT box binding transcription factor.

Place, publisher, year, edition, pages
2006. Vol. 80, no 10, 5010-5020 p.
Keyword [en]
Adenoviridae/*genetics/*metabolism, Carrier Proteins/biosynthesis/genetics/*physiology, Cell Line, DNA Replication/physiology, Humans, Mitochondrial Proteins/biosynthesis/genetics/*physiology, NFI Transcription Factors/genetics/physiology, Phosphorylation, Promoter Regions (Genetics), RNA Polymerase II/antagonists & inhibitors/*metabolism, Recombination; Genetic, Repressor Proteins/biosynthesis/genetics/*physiology, Tetracycline/pharmacology, Transcription; Genetic/*physiology
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-94323DOI: 10.1128/JVI.80.10.5010-5020.2006PubMedID: 16641292OAI: oai:DiVA.org:uu-94323DiVA: diva2:168134
Available from: 2006-04-20 Created: 2006-04-20 Last updated: 2013-03-21Bibliographically approved
In thesis
1. Functional Characterization of the Cellular Protein p32: A Protein Regulating Adenovirus Transcription and Splicing Through Targeting of Phosphorylation
Open this publication in new window or tab >>Functional Characterization of the Cellular Protein p32: A Protein Regulating Adenovirus Transcription and Splicing Through Targeting of Phosphorylation
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cellular processes involved in the conversion of the genetic information from DNA into a protein are often regulated by reversible phosphorylation reactions. By modulating the phosphorylated status of key proteins their activity can either be enhanced or repressed. In this thesis I have studied the significance of phosphorylation in the regulation of transcription and splicing using human adenovirus as a model system.

The results show that the activity of the cellular SR family of splicing enhancer or repressor proteins are reduced in adenovirus infected nuclear extracts by a virus-induced hypophosphorylation. The viral E4-ORF4 was shown to induce SR protein dephosphorylation by recruiting the cellular protein phosphatase PP2A. The E4-ORF4/PP2A complex was shown to relieve the SR protein-mediated repression of late virus-specific splicing and further activate alternative splicing in transiently transfected cells. Collectively, these results showed that alternative splicing, like many other biological processes, is regulated by reversible protein phosphorylation.

Similarly, the cellular p32 protein was shown to cause hypophosphorylation of the SR protein ASF/SF2 resulting in a reduced RNA binding capacity of ASF/SF2. This change in ASF/SF2 RNA binding also had a drastic effect on the function of ASF/SF2 as a regulatory protein affecting splice site choice. The cellular p32 protein and the viral E4-ORF4 protein both target the same cellular splicing factor, ASF/SF2. However, they regulate splicing by different mechanisms. E4-ORF4 recruits a phosphatase to dephosphorylate ASF/SF2, while p32 sequester ASF/SF2 in an inactive complex.

Further, we demonstrated that overexpression of p32 during a lytic infection suppressed transcription from the adenovirus major late transcription unit. p32 induced a selective repression of CAAT-box containing promoters indicating the involvement of the transcription factor CBF/NF-Y in this regulation. A further analysis showed that p32 caused a hyperphosphorylation of the CTD of RNA Pol II, which resulted in a significant reduction in the processivity of Pol II during the elongation phase of transcription.

In summary, we have shown that E4-ORF4 regulates the activity of splicing regulatory SR proteins, and that p32 regulates the activity of the SR protein ASF/SF2 in splicing and Pol II processivity during transcription elongation. Mechanistically, both E4-ORF4 and p32 appears to function by regulating the phosphorylated status of key cellular proteins involved in these processes.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2006. 69 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 139
Cell and molecular biology, Adenovirus, p32, ASF/SF2, E4-ORF4, Splicing, Transcription, CTD, RNA Pol II, Cell- och molekylärbiologi
urn:nbn:se:uu:diva-6794 (URN)91-554-6533-1 (ISBN)
Public defence
2006-05-11, C10:301, BMC, Husargatan 3, Uppsala, 13:15
Available from: 2006-04-20 Created: 2006-04-20Bibliographically approved

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Akusjärvi, Göran
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