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Multiple splicing enhancers and hnRNPA1-binding silencers control the HPV-16 late 3' splice site
Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
In: Journal of VirologyArticle in journal (Refereed) Submitted
URN: urn:nbn:se:uu:diva-93430OAI: oai:DiVA.org:uu-93430DiVA: diva2:166901
Available from: 2005-09-19 Created: 2005-09-19Bibliographically approved
In thesis
1. Regulation of Human Papillomavirus Type 16 mRNA Splicing and Polyadenylation
Open this publication in new window or tab >>Regulation of Human Papillomavirus Type 16 mRNA Splicing and Polyadenylation
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Human papillomavirus type 16 (HPV-16) is the major causative agent of cervical cancer. The life cycle of this oncogenic DNA tumour virus is strictly associated with the differentiation program of the infected epithelial cells. Expression of the viral capsid genes L1 and L2 can only be detected in the terminally differentiated epithelial cells. The studies here focus on the regulation of HPV-16 late gene expression, which is under tight regulation.

Our experimental system consisted of almost the full length HPV-16 genome driven by a strong CMV promoter. This plasmid and mutants thereof could be transfected into HeLa cells and RNA levels monitored. Using this system, we identified an hnRNP A1-dependent splicing silencer between positions 178 and 226 of the L1 gene. This silencer inhibited the use of the 3' splice site, located immediately upstream of the L1 AUG. We speculate that this splicing silencer plays an essential role in preventing late gene expression at an early stage of the viral life cycle. We subsequently identified a splicing enhancer located in the first 17 nucleotides of L1 that may be needed to counteract the multiple hnRNP A1 dependent splicing silencers in the L1 coding region. A 55kDa protein specifically bound to this splicing enhancer. We also demonstrated that binding of the cellular factors to the splicing silencer in the L1 coding region had an inhibitory effect on expression from L1 cDNA expression plasmids.

The HPV-16 genome is divided into the early region and the late region, separated by the early poly(A) signal (pAE). pAE is used preferentially early in infection, thereby efficiently blocking late gene expression. We demonstrated that a 57 nucleotide U-rich region of the early 3’untranslated region (3’eUTR) acted as an enhancing upstream element on the usage of pAE. We demonstrated that this U-rich region specifically interacts with hFip1, CstF-64, hnRNP C1/C2 and PTB, suggesting that these factors were either enhancing or regulating polyadenylation at the HPV-16 pAE.

In conclusion, two regulatory RNA elements that both act to prevent late gene expression at an early stage in the viral life cycle and in proliferating cells were identified: a splicing silencer in the late region and an upstream u-rich element at the pAE.

Place, publisher, year, edition, pages
Uppsala: Institutionen för medicinsk biokemi och mikrobiologi, 2005. 58 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 67
Medical sciences, human papillomavirus type 16, gene regulation, splicing, polyadenylation, 3' UTR, MEDICIN OCH VÅRD
National Category
Medical and Health Sciences
urn:nbn:se:uu:diva-5919 (URN)91-554-6328-2 (ISBN)
Public defence
2005-10-06, C10:305, Biomedical Center, Husargatan 3, Uppsala, 10:30
Available from: 2005-09-19 Created: 2005-09-19 Last updated: 2013-09-20Bibliographically approved

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