Suppression of HPV-16 late L1 5′-splice site SD3632 by binding of hnRNP D proteins and hnRNP A2/B1 to upstream AUAGUA RNA motifs

Li, Xiaoze; Johansson, Cecilia; Glahder, Jacob; Mossberg, Ann-Kristin; Schwartz, Stefan

doi:10.1093/nar/gkt803

Cited by 42 publications

(92 citation statements)

References 72 publications

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“…These viral RNA cis elements (ESE and ESS) were originally discovered in bovine papillomavirus late pre-mRNAs (4,5,8,9) and later in HPV16 pre-mRNAs (12,14,21,22). In general, the ESE and ESS are bipartite elements (4,49), commonly located downstream of a suboptimal 3= splice site to regulate selection of an alternative upstream splice site by interaction with various cellular splicing factors (5,6,11,37,48,(50)(51)(52).…”

Section: Discussionmentioning

confidence: 99%

“…Alternative RNA splicing of the HPV polycistronic pre-mRNAs plays a crucial role in regulation of viral gene expression (2,3). Although the molecular mechanisms that regulate alternative RNA splicing of bovine papillomavirus type 1 (BPV-1) (4-11) and HPV16 pre-mRNA transcripts (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22) have been extensively studied in the past, a full transcription map of HPV18 in productive infection, the second most prevalent high-risk HPV genotype in association with cervical cancer (23), was constructed only recently (24), and the mechanistic regulation of HPV18 RNA splicing remains poorly investigated. HPV18 pre-mRNAs are transcribed mainly from a major early promoter, P 55/102 , or a major late promoter, P 811 , although a few other, weak promoters exist in the virus genome (24,25).…”

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confidence: 99%

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Serine/Arginine-Rich Splicing Factor 3 and Heterogeneous Nuclear Ribonucleoprotein A1 Regulate Alternative RNA Splicing and Gene Expression of Human Papillomavirus 18 through Two Functionally Distinguishable cis Elements

Ajiro

Tang

Doorbar

et al. 2016

J Virol

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Human papillomavirus 18 (HPV18) is the second most common oncogenic HPV type associated with cervical, anogenital, and oropharyngeal cancers. Like other oncogenic HPVs, HPV18 encodes two major (one early and one late) polycistronic premRNAs that are regulated by alternative RNA splicing to produce a repertoire of viral transcripts for the expression of individual viral genes. However, RNA cis-regulatory elements and trans-acting factors contributing to HPV18 alternative RNA splicing remain unknown. In this study, an exonic splicing enhancer (ESE) in the nucleotide (nt) 3520 to 3550 region in the HPV18 genome was identified and characterized for promotion of HPV18 929^3434 splicing and E1^E4 production through interaction with SRSF3, a host oncogenic splicing factor differentially expressed in epithelial cells and keratinocytes. Introduction of point mutations in the SRSF3-binding site or knockdown of SRSF3 expression in cells reduces 929^3434 splicing and E1^E4 production but activates other, minor 929^3465 and 929^3506 splicing. Knockdown of SRSF3 expression also enhances the expression of E2 and L1 mRNAs. An exonic splicing silencer (ESS) in the HPV18 nt 612 to 639 region was identified as being inhibitory to the 233^416 splicing of HPV18 E6E7 pre-mRNAs via binding to hnRNP A1, a well-characterized, abundantly and ubiquitously expressed RNA-binding protein. Introduction of point mutations into the hnRNP A1-binding site or knockdown of hnRNP A1 expression promoted 233^416 splicing and reduced E6 expression. These data provide the first evidence that the alternative RNA splicing of HPV18 pre-mRNAs is subject to regulation by viral RNA cis elements and host trans-acting splicing factors. IMPORTANCEExpression of HPV18 genes is regulated by alternative RNA splicing of viral polycistronic pre-mRNAs to produce a repertoire of viral early and late transcripts. RNA cis elements and trans-acting factors contributing to HPV18 alternative RNA splicing have been discovered in this study for the first time. The identified ESS at the E7 open reading frame (ORF) prevents HPV18 233^416 splicing in the E6 ORF through interaction with a host splicing factor, hnRNP A1, and regulates E6 and E7 expression of the early E6E7 polycistronic pre-mRNA. The identified ESE at the E1^E4 ORF promotes HPV18 929^3434 splicing of both viral early and late pre-mRNAs and E1^E4 production through interaction with SRSF3. This study provides important observations on how alternative RNA splicing of HPV18 pre-mRNAs is subject to regulation by viral RNA cis elements and host splicing factors and offers potential therapeutic targets to overcome HPV-related cancer.H igh-risk human papillomavirus (HPV) is associated with more than 95% of cervical cancer, 40 to 90% of anogenital cancer, and 10 to 60% of oropharyngeal cancer with geographic variation (1). Two major polycistronic pre-mRNAs, early and late pre-mRNAs, are transcribed from the high-risk HPV genome. Alternative RNA splicing of the HPV polycistronic pre-mRNAs plays a crucial role in regu...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Serine/Arginine-Rich Splicing Factor 3 and Heterogeneous Nuclear Ribonucleoprotein A1 Regulate Alternative RNA Splicing and Gene Expression of Human Papillomavirus 18 through Two Functionally Distinguishable cis Elements

Ajiro

Tang

Doorbar

et al. 2016

J Virol

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“…Plasmids-The following plasmids have been described previously: pCL086 (32), pBEL (29), pBELDUTR (18), pBELsLuc (28), pBELMsLuc (25,28), pRSVneo (25,28), pBSpD1MCAT (28), p4xATAGTA (28), p4xMUT (28), and pHPV16ANSL (25,28). We thank Andras Nagy (University of Toronto) for providing pCAGGS-nlscre (33).…”

Section: Methodsmentioning

confidence: 99%

“…Propagation and Transfection of Human Primary Keratinocytes-Propagation of neonatal human epidermal keratinocytes has been described previously (28). Briefly, neonatal human epidermal keratinocyte cells were purchased from Gibco and were propagated in EpiLife medium supplemented with human keratinocyte growth supplement (Gibco).…”

Section: Methodsmentioning

confidence: 99%

“…HPV16 late 5Ј-splice site SD3632 is used only for production of spliced late L1 mRNAs (15,16). In undiffer-entiated cells, SD3632 is suppressed by upstream sequences that encode two AUAGUA motifs and interact specifically with members of the hnRNP D family and hnRNP A2/B1 (28). HPV16 late slice site SD3632 is spliced to late 3Ј-splice site SA5639 located immediately upstream of the L1 AUG. in mitotic cells, SA5639 is suppressed.…”

Section: Human Papillomavirus (Hpv)mentioning

confidence: 99%

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Heterogeneous Nuclear Ribonucleoprotein C Proteins Interact with the Human Papillomavirus Type 16 (HPV16) Early 3′-Untranslated Region and Alleviate Suppression of HPV16 Late L1 mRNA Splicing

Dhanjal¹,

Kajitani²,

Glahder

et al. 2015

Journal of Biological Chemistry

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Background: HPV16 late gene expression is suppressed in mitotic cells. Results: hnRNP C proteins bind to the HPV16 early, untranslated region and activate HPV16 late mRNA splicing. Conclusion: hnRNP C proteins control HPV16 late gene expression. Significance: hnRNP C proteins may contribute to the ability of HPV16 to avoid the immune system and establish persistent infections that progress to cancer.

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Short half‐life of HPV16 E6 and E7 mRNAs sensitizes HPV16‐positive tonsillar cancer cell line HN26 to DNA‐damaging drugs

Nilsson

Zheng

et al. 2018

Intl Journal of Cancer

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Here we show that treatment of the HPV16‐positive tonsillar cancer cell line HN26 with DNA alkylating cancer drug melphalan‐induced p53 and activated apoptosis. Melphalan reduced the levels of RNA polymerase II and cellular transcription factor Sp1 that were associated with HPV16 DNA. The resulting inhibition of transcription caused a rapid loss of the HPV16 early mRNAs encoding E6 and E7 as a result of their inherent instability. As a consequence of HPV16 E6 and E7 down‐regulation, the DNA damage inflicted on the cells by melphalan caused induction of p53 and activation of apoptosis in the HN26 cells. The BARD1‐negative phenotype of the HN26 cells may have contributed to the failure to repair DNA damage caused by melphalan, as well as to the efficient apoptosis induction. Finally, nude mice carrying the HPV16 positive tonsillar cancer cells responded better to melphalan than to cisplatin, the chemotherapeutic drug of choice for tonsillar cancer. We concluded that the short half‐life of the HPV16 E6 and E7 mRNAs renders HPV16‐driven tonsillar cancer cells particularly sensitive to DNA damaging agents such as melphalan since melphalan both inhibits transcription and causes DNA damage.

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Suppression of HPV-16 late L1 5′-splice site SD3632 by binding of hnRNP D proteins and hnRNP A2/B1 to upstream AUAGUA RNA motifs

Cited by 42 publications

References 72 publications

Serine/Arginine-Rich Splicing Factor 3 and Heterogeneous Nuclear Ribonucleoprotein A1 Regulate Alternative RNA Splicing and Gene Expression of Human Papillomavirus 18 through Two Functionally Distinguishable cis Elements

Serine/Arginine-Rich Splicing Factor 3 and Heterogeneous Nuclear Ribonucleoprotein A1 Regulate Alternative RNA Splicing and Gene Expression of Human Papillomavirus 18 through Two Functionally Distinguishable cis Elements

Heterogeneous Nuclear Ribonucleoprotein C Proteins Interact with the Human Papillomavirus Type 16 (HPV16) Early 3′-Untranslated Region and Alleviate Suppression of HPV16 Late L1 mRNA Splicing

Short half‐life of HPV16 E6 and E7 mRNAs sensitizes HPV16‐positive tonsillar cancer cell line HN26 to DNA‐damaging drugs

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