Alternative splicing of human papillomavirus type-16 E6/E6* early mRNA is coupled to EGF signaling via Erk1/2 activation

Abstract

Certain types of human papillomaviruses (HPVs) are etiologically linked to cervical cancer. Their transforming capacity is encoded by a polycistronic premRNA, where alternative splicing leads to the translation of functional distinct proteins such as E6, E6*, and E7. Here we show that splicing of HPV16 E6/E7 ORF cassette is regulated by the epidermal growth factor (EGF) pathway. The presence of EGF was coupled to preferential E6 expression, whereas depletion of EGF, or treatment with EGF receptor (EGFR) neutralizing antibodies or the EGFR inhibitor tyrphostin AG1478, resulted in E6 exon exclusion in favor of E6*. As a consequence, increased p53 levels and enhanced translation of E7 with a subsequent reduction of the retinoblastoma protein pRb could be discerned. E6 exon exclusion upon EGF depletion was independent from promoter usage, mRNA stability, or selective mRNA transport. Time-course experiments and incubation with cycloheximide demonstrated that E6 alternative splicing is a direct and reversible effect of EGF signal transduction, not depending on de novo protein synthesis. Within this process, Erk1/2-kinase activation was the critical event for E6 exon inclusion, mediated by the upstream MAP kinase MEK1/2. Moreover, siRNA knockdown experiments revealed an involvement of splicing factors hnRNPA1 and hnRNPA2 in E6 exon exclusion, whereas the splicing factors Brm and Sam68 were found to promote E6 exon inclusion. Because there is a natural gradient of EGF and EGF receptor expression in the stratified epithelium, it is reasonable to assume that EGF modulates E6/E7 splicing during the viral life cycle and transformation.P articular types of human papillomaviruses (HPVs) such as HPV16, 18, 31, and 33, respectively, are the etiological agents for the development of anogenital tumors. Their transforming potential is encoded by the viral oncoproteins E6 and E7, where among other functions, E6 labilizes p53 and prevents apoptosis, and E7 promotes cell cycle progression by degrading the retinoblastoma protein pRb (1). HPV gene transcription is regulated by two main promoters, the early p97 and the late p670 promoter (2). Activation of either promoter is regulated by differentiation, resulting in the synthesis of polycistronic mRNAs, which are further regulated by differential splicing (3, 4). For polycistronic mRNAs, it is known that only the first ORF is translated efficiently when intercistronic distances are short (5).Alternative splicing can be regulated dependent on the developmental stage or by extracellular stimuli, where an increasing number of pathways and splicing factors have been identified (6). For splicing reaction, the spliceosome recognizes exon-intron boundaries of the 5′-donor and 3′-acceptor splice site. Furthermore, specific sequence motifs within exons can positively or negatively influence the recognition of nearby splice sites (7). The activity of so-called exonic splicing enhancers or exonic splicing silencers can be modulated by splicing factors like SR proteins or hnRNPs, which in turn all...

The RNA-binding protein Sam68 is a multifunctional player in human cancer

“…The functional interaction between the MAPK pathway and the activity of the Brm/Sam68 complex is also involved in the splicing of the human papillomavirus (HPV) polycistronic pre-mRNA. It was shown that EGF stimulated splicing of the E6 variant of HPV16, through a mechanism depending on activation of ERK1/2 and Brm/Sam68 (Rosenberger et al 2010). This observation suggests the involvement of Sam68 in the regulation of the life cycle of HPV and neoplastic transformation in cervical cancer.…”

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

“…In HPV16, hnRNP A1 binds to an ESS in the L1 coding region and regulates HPV16 L1 RNA splicing (12,22). hnRNP A1 and A2 together were also proposed to regulate HPV16 E6 intron splicing (226^409 splicing) (19), but the mechanism of this regulation remains unknown.…”

“…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).…”

EGFR Promoter Methylation Detection in Cervical Cancer by a Hybridization-Fluorescence Polarization Assay

“…HPV E5 protein mediates its carcinogenic effects using EGFR-dependent processes. [23][24][25][26] EGFR-directed therapies are effective treatments for some cancers associated with HPV infection. Reasons for the absence of a major clinical breakthrough in some patients may be multiple.…”