title: Human Papillomavirus 16 Oncoprotein Expression Is Controlled by the Cellular Splicing Factor SRSF2 (SC35).
abstract: High-risk human papillomaviruses (HR-HPV) cause anogenital cancers, including cervical cancer, and head and neck cancers. Human papillomavirus 16 (HPV16) is the most prevalent HR-HPV. HPV oncogenesis is driven by two viral oncoproteins, E6 and E7, which are expressed through alternative splicing of a polycistronic RNA to yield four major splice isoforms (E6 full length, E6*I, E6*II, E6*X). The production of multiple mRNA isoforms from a single gene is controlled by serine/arginine-rich splicing factors (SRSFs), and HPV16 infection induces overexpression of a subset of these, SRSFs 1, 2, and 3. In this study, we examined whether these proteins could control HPV16 oncoprotein expression. Small interfering RNA (siRNA) depletion experiments revealed that SRSF1 did not affect oncoprotein RNA levels. While SRSF3 knockdown caused some reduction in E6E7 expression, depletion of SRSF2 resulted in a significant loss of E6E7 RNAs, resulting in reduced levels of the E6-regulated p53 proteins and E7 oncoprotein itself. SRSF2 contributed to the tumor phenotype of HPV16-positive cervical cancer cells, as its depletion resulted in decreased cell proliferation, reduced colony formation, and increased apoptosis. SRSF2 did not affect transcription from the P97 promoter that controls viral oncoprotein expression. Rather, RNA decay experiments showed that SRSF2 is required to maintain stability of E6E7 mRNAs. These data show that SRSF2 is a key regulator of HPV16 oncoprotein expression and cervical tumor maintenance.
  
  
  OBJECTIVE
  Expression of the HPV16 oncoproteins E7 and E6 drives HPV-associated tumor formation. Although increased transcription may yield increased levels of E6E7 mRNAs, it is known that the RNAs can have increased stability upon integration into the host genome. SR splicing factors (SRSFs) control splicing but can also control other events in the RNA life cycle, including RNA stability. Previously, we demonstrated increased levels of SRSFs 1, 2, and 3 during cervical tumor progression. Now we show that SRSF2 is required for expression of E6E7 mRNAs in cervical tumor but not nontumor cells and may act by inhibiting their decay. SRSF2 depletion in W12 tumor cells resulted in increased apoptosis, decreased proliferation, and decreased colony formation, suggesting that SRSF2 has oncogenic functions in cervical tumor progression. SRSF function can be targeted by known drugs that inhibit SRSF phosphorylation, suggesting a possible new avenue in abrogating HPV oncoprotein activity.
authors.affiliation: MRC-University of Glasgow Centre for Virus Research, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom.
authors.forename: Melanie
authors.initials: M
authors.lastname: McFarlane
authors.affiliation: MRC-University of Glasgow Centre for Virus Research, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom.
authors.forename: Alasdair I
authors.initials: AI
authors.lastname: MacDonald
authors.affiliation: MRC-University of Glasgow Centre for Virus Research, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom.
authors.forename: Andrew
authors.initials: A
authors.lastname: Stevenson
authors.affiliation: MRC-University of Glasgow Centre for Virus Research, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom Sheila.Graham@gla.ac.uk.
authors.forename: Sheila V
authors.initials: SV
authors.lastname: Graham
mesh_terms: D017398:Alternative Splicing; D017209:Apoptosis; D002460:Cell Line; D045744:Cell Line, Tumor; D005260:Female; D015967:Gene Expression Regulation, Viral; D055785:Gene Knockdown Techniques; D005814:Genes, Viral; D054884:Host-Pathogen Interactions; D052162:Human papillomavirus 16 / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D050725:Papillomavirus E7 Proteins / Q000235:genetics*; D030361:Papillomavirus Infections / Q000378:metabolism / Q000821:virology; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics*; D012261:Ribonucleoproteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000068103:Serine-Arginine Splicing Factors; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D002583:Uterine Cervical Neoplasms / Q000235:genetics / Q000378:metabolism / Q000821:virology
chemical_list: C068651:E6 protein, Human papillomavirus type 16; D009687:Nuclear Proteins; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D012333:RNA, Messenger; D012367:RNA, Viral; D012097:Repressor Proteins; D012261:Ribonucleoproteins; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; C059731:oncogene protein E7, Human papillomavirus type 16; C073288:SRSF2 protein, human; D000068103:Serine-Arginine Splicing Factors