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A 57-nucleotide upstream early polyadenylation element in human papillomavirus type 16 interacts with hFip1, CstF-64, hnRNP C1/C2, and polypyrimidine tract binding protein
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.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
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2005 (English)In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 79, no 7, 4270-4288 p.Article in journal (Refereed) Published
Abstract [en]

We have investigated the role of the human papillomavirus type 16 (HPV-16) early untranslated region (3' UTR) in HPV-16 gene expression. We found that deletion of the early 3' UTR reduced the utilization of the early polyadenylation signal and, as a consequence, resulted in read-through into the late region and production of late L1 and L2 mRNAs. Deletion of the U-rich 3' half of the early 3' UTR had a similar effect, demonstrating that the 57-nucleotide U-rich region acted as an enhancing upstream element on the early polyadenylation signal. In accordance with this, the newly identified hFip1 protein, which has been shown to enhance polyadenylation through U-rich upstream elements, interacted specifically with the HPV-16 upstream element. This upstream element also interacted specifically with CstF-64, hnRNP C1/C2, and polypyrimidine tract binding protein, suggesting that these factors were either enhancing or regulating polyadenylation at the HPV-16 early polyadenylation signal. Mutational inactivation of the early polyadenylation signal also resulted in increased late mRNA production. However, the effect was reduced by the activation of upstream cryptic polyadenylation signals, demonstrating the presence of additional strong RNA elements downstream of the early polyadenylation signal that direct cleavage and polyadenylation to this region of the HPV-16 genome. In addition, we identified a 3' splice site at genomic position 742 in the early region with the potential to produce E1 and E4 mRNAs on which the E1 and E4 open reading frames are preceded only by the suboptimal E6 AUG. These mRNAs would therefore be more efficiently translated into E1 and E4 than previously described HPV-16 E1 and E4 mRNAs on which E1 and E4 are preceded by both E6 and E7 AUGs.

Place, publisher, year, edition, pages
2005. Vol. 79, no 7, 4270-4288 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-93537DOI: 10.1128/JVI.79.7.4270-4288.2005PubMedID: 15767428OAI: oai:DiVA.org:uu-93537DiVA: diva2:167044
Available from: 2005-10-06 Created: 2005-10-06 Last updated: 2013-09-20Bibliographically approved
In thesis
1. Regulation of RNA Processing in Human Papillomavirus Type 16
Open this publication in new window or tab >>Regulation of RNA Processing in Human Papillomavirus Type 16
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Human papillomavirus type 16 (HPV-16) is the major cause of cervical cancer. HPV-16 gene expression is tightly linked to the differentiation programme of the infected epithelium. Expression of the late genes, L1 and L2, encoding the capsid proteins, is delayed until the more terminally differentiated cells. Successful inhibition of HPV-16 late gene expression early in the viral life cycle is essential for persistence of infection, the highest risk factor for cervical cancer.

The goal of this thesis was to identify regulatory RNA elements and cellular factors that influence RNA processing events, such as alternative splicing and polyadenylation, during late gene expression. For this purpose, transfection of plasmids containing almost the full-length HPV-16 genome into HeLa cells, followed by RNA analysis, was employed. An exonic splicing enhancer (ESE) was identified that firmly supported the use of the E4 3’ splice site. A key regulator of HPV-16 gene expression, the E4 ESE was required for early mRNA splicing and polyadenylation, as well as for inhibition of premature late gene expression. The early polyadenylation signal (pAE) is also an important block of premature late gene expression. An upstream polyadenylation element (USE) was identified in the early 3’ untranslated region that enhanced polyadenylation at pAE, and interacted specifically with the cellular factors CstF-64, hnRNP C1/C2, PTB and hFip1. With the help of adenoviral E4orf4, a protein which causes dephosphorylation of SR proteins, we found that overexpression of SRp30c activated HPV-16 late gene expression by an exon skipping mechanism, and that SRp30c may interfere with early mRNA terminal exon definition.

This work identified a crucial splicing enhancer, as well as a number of cellular proteins binding to an USE in the early region of HPV-16. Furthermore, the cellular splicing factor SRp30c was shown to play a role in the regulation of HPV-16 late gene expression.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2005. 77 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 75
Keyword
Molecular biology, RNA processing, human papillomavirus, HPV-16, alternative splicing, polyadenylation, exonic splicing enhancer, 3'UTR, upstream polyadenylation element, SR proteins, Molekylärbiologi
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-5972 (URN)91-554-6356-8 (ISBN)
Public defence
2005-10-27, Room B42, BMC, Husargatan 3, Uppsala, 10:00
Opponent
Supervisors
Available from: 2005-10-06 Created: 2005-10-06Bibliographically approved
2. 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.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 67
Keyword
Medical sciences, human papillomavirus type 16, gene regulation, splicing, polyadenylation, 3' UTR, MEDICIN OCH VÅRD
National Category
Medical and Health Sciences
Identifiers
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
Opponent
Supervisors
Available from: 2005-09-19 Created: 2005-09-19 Last updated: 2013-09-20Bibliographically approved

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Öberg, DanielSchwartz, Stefan

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