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

Abstract

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...

Host control of human papillomavirus infection and disease

“…In the case of the high-risk HPV types, which are contained in the Alpha genus, this is a largely E6-mediated function. HPV16 E6 gene expression is facilitated in the growth factor rich environment of the basal and parabasal epithelial layers, with an increase in E6 abundance driving a corresponding decrease in p53 activity as a result of E6-mediated proteasome degradation (9,10). In the epithelial basal layer, this p53 reduction leads to a loss of p53 transcriptional activity, which in turn leads to reduced levels of the Notch receptor on the cell surface.…”

The Role of E6 Spliced Isoforms (E6*) in Human Papillomavirus-Induced Carcinogenesis

“…The splicing pattern of HPV type 16 has been thoroughly studied and the following spliced transcripts have been identified: E6*I, E6*II, E6*III, E6^E7, E6^E7*I, E6^E7*II, E6*IV, E6*V and E6*VI [ 18 , 59 , 60 , 61 , 62 ]. Conversely, the described transcripts for HPV18 are: E6*I, E6*II, E6*III, E6^E7 [ 60 , 63 , 64 ]. Less is known about transcripts resulting from splicing in the E6 pre-mRNA of other HR-HPV types, such as HPV31 having E6*I and E6^E4; HPV33 with E6*I, E6*II and E6*III; and HPV58 with E6*I and E6*II [ 65 , 66 , 67 , 68 ].…”

“…In contrast to hnRNPA1 only, that in the presence of Epidermal Growth Factor (EGF) induces an increase in un-spliced E6 mRNA [ 98 ]. This evidence could be associated to the exonic splicing silencer (ESS) within the E7 ORF, which contains an hnRNPA1 binding motif that reduces 233^416 splicing (E6*I) and induces E6 expression in HPV18-transfected or -infected cells [ 64 ].…”

Role of Viral Ribonucleoproteins in Human Papillomavirus Type 16 Gene Expression

“…The control of these splice sites by cis-acting sequences and trans-acting factors is currently under investigation, and hitherto it has been reported that 226^409 splicing in HPV16 and the corresponding splicing event in HPV 18 (233^416) are negatively regulated by hnRNP A1 that binds to exonic splicing silencers located in the E7-coding region [ 56 , 57 ] ( Figure 5 ). This suppression promotes production of unspliced E6 mRNAs at the expense of the spliced E7 mRNAs and highlights the importance of hnRNP A1 in the control of expression of the two oncogenes E6 and E7.…”

“…Despite these properties, it conforms poorly to the consensus 3′-splice site ( Table 1 ). The splicing enhancer immediately downstream of SA3358 is present in both HPV16 and HPV18 and interacts with SR proteins (SRSF1, SRSF3, and SRSF9) ( Figure 4 ) [ 56 , 58 , 59 , 60 , 61 , 62 , 63 ]. The sequences adjacent to SA3358 also constitute hot spots for multiple RNA-binding proteins, underscoring the complexity of this region and the control of SA3358 ( Figure 3 and Figure 5 ) [ 48 ].…”