Multiple ASF/SF2 sites in the human papillomavirus type 16 (HPV-16) E4-coding region promote splicing to the most commonly used 3'-splice site on the HPV-16 genome.
Our results presented here demonstrate that the most abundant human papillomavirus type 16 (HPV-16) mRNAs expressing the viral oncogenes E6 and E7 are regulated by cellular ASF/SF2, itself defined as a proto-oncogene and overexpressed in cervical cancer cells. We show that the most frequently used 3'-splice site on the HPV-16 genome, site SA3358, which is used to produce primarily E4, E6, and E7 mRNAs, is regulated by ASF/SF2. Splice site SA3358 is immediately followed by 15 potential binding sites for the splicing factor ASF/SF2. Recombinant ASF/SF2 binds to the cluster of ASF/SF2 sites. Mutational inactivation of all 15 sites abolished splicing to SA3358 and redirected splicing to the downstream-located, late 3'-splice site SA5639. Overexpression of a mutant ASF/SF2 protein that lacks the RS domain, also totally inhibited the usage of SA3358 and redirected splicing to the late 3'-splice site SA5639. The 15 ASF/SF2 binding sites could be replaced by an ASF/SF2-dependent, HIV-1-derived splicing enhancer named GAR. This enhancer was also inhibited by the mutant ASF/SF2 protein that lacks the RS domain. Finally, silencer RNA (siRNA)-mediated knockdown of ASF/SF2 caused a reduction in spliced HPV-16 mRNA levels. Taken together, our results demonstrate that the major HPV-16 3'-splice site SA3358 is dependent on ASF/SF2. SA3358 is used by the most abundantly expressed HPV-16 mRNAs, including those encoding E6 and E7. High levels of ASF/SF2 may therefore be a requirement for progression to cervical cancer. This is supported by our earlier findings that ASF/SF2 is overexpressed in high-grade cervical lesions and cervical cancer.
Somberg
Monika
M
Department of Medical Biochemistry and Microbiology, BMC, Uppsala University, Sweden.
Schwartz
Stefan
S
D001483:Base Sequence; D015967:Gene Expression Regulation, Viral; D006367:HeLa Cells; D054884:Host-Pathogen Interactions; D052162:Human papillomavirus 16 / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D000090063:Proto-Oncogene Mas; D022821:RNA Splice Sites; D012326:RNA Splicing; D012367:RNA, Viral / Q000378:metabolism*; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors
C000715470:MAS1 protein, human; D009687:Nuclear Proteins; D000090063:Proto-Oncogene Mas; D022821:RNA Splice Sites; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors