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The role of cellular RNA processing functions in Human papillomavirus type 16 gene regulation

Nilsson, Kersti LU (2019) In Lund University, Faculty of Medicine Doctoral Dissertation Series 2019(92).
Abstract
Infections with Human papillomavirus type 16 (HPV16) is the most the most common, sexually transferred and is responsible for genital warts, cervical cancers and a growing number of head and neck cancers. Knowledge about how HPV16 interacts with the infected cell to regulate its gene expression is essential for therapeutic development.

Here, we show that the viral E5 protein which may contribute to carcinogenesis can only be expressed efficiently during the early infection. E5 is the last open reading frame (ORF) on the early pre-mRNAs. Therefore, its expression requires removal of upstream, inhibitory E7 and E1 ORFs by alternative splicing.

Alternative splicing is also required for HPV16 later gene expression together... (More)
Infections with Human papillomavirus type 16 (HPV16) is the most the most common, sexually transferred and is responsible for genital warts, cervical cancers and a growing number of head and neck cancers. Knowledge about how HPV16 interacts with the infected cell to regulate its gene expression is essential for therapeutic development.

Here, we show that the viral E5 protein which may contribute to carcinogenesis can only be expressed efficiently during the early infection. E5 is the last open reading frame (ORF) on the early pre-mRNAs. Therefore, its expression requires removal of upstream, inhibitory E7 and E1 ORFs by alternative splicing.

Alternative splicing is also required for HPV16 later gene expression together with an inhibition of the early polyadenylation signal. We report that induction of the DNA damage response (DDR) by alkylating agent melphalan caused both an activation of late gene splicing and inhibition of the early polyadenylation signal. The connection between induction of DDR and HPV16 late gene expression by melphalan was dependent on ATM signaling and caused an accumulation of phosphorylated BRCA1 on HPV16 DNA. BRAC1 also interacted with splice factors U2AF65 and hnRNPC that were recruited to HPV16 mRNAs to generate a expression of late, L1 and L2 HPV16 mRNAs. The ATM-branch of the DDR is hijacked by HPV for productive viral replication prior to late gene expression. Therefore, we suggest that HPV16 also has evolved to utilize the connection between DDR and RNA-processing for late gene activation.

Finally, we investigated the presence of methylated N6-adenosine (m6A) on HPV16 mRNAs in connection to regulation of alternative splicing. Here, we report that HPV16 mRNAs undergoes methylation and that the proteins associated with m6A affects viral splicing. In particular, splicing of mRNAs that express the viral oncoproteins E6 and E7 were affected in a mutually exclusive manner. The same pattern was also observed for mRNAs expressing viral replication proteins E1 and E2. Further investigations will reveal the true regulatory importance of m6A for splicing of these mRNAs. In conclusion, this thesis identifies the main E5 mRNA, connects HPV16 late gene expression to the DDR and suggests a regulatory function of m6A in HPV16 alternative splicing.
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author
supervisor
opponent
  • docent Gekara, Nelson, Umeå University
organization
publishing date
type
Thesis
publication status
published
subject
keywords
RNA Splicing; Polyadenylation; DNA damage response, Human papillomavirus; Influenza A
in
Lund University, Faculty of Medicine Doctoral Dissertation Series
volume
2019
issue
92
pages
64 pages
publisher
Lund University: Faculty of Medicine
defense location
BMC I1345, Sölvegatan 19 i Lund
defense date
2019-10-19 09:00:00
ISSN
1652-8220
ISBN
978-91-7619-821-6
language
English
LU publication?
yes
id
caa4cff4-3dc2-4659-bd09-8e97ce6e6511
date added to LUP
2019-09-30 15:38:13
date last changed
2021-04-16 10:44:17
@phdthesis{caa4cff4-3dc2-4659-bd09-8e97ce6e6511,
  abstract     = {{Infections with Human papillomavirus type 16 (HPV16) is the most the most common, sexually transferred and is responsible for genital warts, cervical cancers and a growing number of head and neck cancers. Knowledge about how HPV16 interacts with the infected cell to regulate its gene expression is essential for therapeutic development. <br/><br/>Here, we show that the viral E5 protein which may contribute to carcinogenesis can only be expressed efficiently during the early infection. E5 is the last open reading frame (ORF) on the early pre-mRNAs. Therefore, its expression requires removal of upstream, inhibitory E7 and E1 ORFs by alternative splicing. <br/><br/>Alternative splicing is also required for HPV16 later gene expression together with an inhibition of the early polyadenylation signal. We report that induction of the DNA damage response (DDR) by alkylating agent melphalan caused both an activation of late gene splicing and inhibition of the early polyadenylation signal. The connection between induction of DDR and HPV16 late gene expression by melphalan was dependent on ATM signaling and caused an accumulation of phosphorylated BRCA1 on HPV16 DNA. BRAC1 also interacted with splice factors U2AF65 and hnRNPC that were recruited to HPV16 mRNAs to generate a expression of late, L1 and L2 HPV16 mRNAs. The ATM-branch of the DDR is hijacked by HPV for productive viral replication prior to late gene expression. Therefore, we suggest that HPV16 also has evolved to utilize the connection between DDR and RNA-processing for late gene activation. <br/><br/>Finally, we investigated the presence of methylated N6-adenosine (m6A) on HPV16 mRNAs in connection to regulation of alternative splicing. Here, we report that HPV16 mRNAs undergoes methylation and that the proteins associated with m6A affects viral splicing. In particular, splicing of mRNAs that express the viral oncoproteins E6 and E7 were affected in a mutually exclusive manner. The same pattern was also observed for mRNAs expressing viral replication proteins E1 and E2. Further investigations will reveal the true regulatory importance of m6A for splicing of these mRNAs. In conclusion, this thesis identifies the main E5 mRNA, connects HPV16 late gene expression to the DDR and suggests a regulatory function of m6A in HPV16 alternative splicing. <br/>}},
  author       = {{Nilsson, Kersti}},
  isbn         = {{978-91-7619-821-6}},
  issn         = {{1652-8220}},
  keywords     = {{RNA Splicing; Polyadenylation; DNA damage response, Human papillomavirus; Influenza A}},
  language     = {{eng}},
  number       = {{92}},
  publisher    = {{Lund University: Faculty of Medicine}},
  school       = {{Lund University}},
  series       = {{Lund University, Faculty of Medicine Doctoral Dissertation Series}},
  title        = {{The role of cellular RNA processing functions in Human papillomavirus type 16 gene regulation}},
  url          = {{https://lup.lub.lu.se/search/files/70160736/kappa.pdf}},
  volume       = {{2019}},
  year         = {{2019}},
}