Exonic splicing enhancer-dependent selection of the bovine papillomavirus type 1 nucleotide 3225 3' splice site can be rescued in a cell lacking splicing factor ASF/SF2 through activation of the phosphatidylinositol 3-kinase/Akt pathway

Bovine papillomavirus type 1 (BPV-1) late pre-mRNAs are spliced in keratinocytes in a differentiation-specific manner: the late leader 5' splice site alternatively splices to a proximal 3' splice site (at nucleotide 3225) to express L2 or to a distal 3' splice site (at nucleotide 3605) to express L1. Two exonic splicing enhancers, each containing two ASF/SF2 (alternative splicing factor/splicing factor 2) binding sites, are located between the two 3' splice sites and have been identified as regulating alternative 3' splice site usage. The present report demonstrates for the first time that ASF/SF2 is required under physiological conditions for the expression of BPV-1 late RNAs and for selection of the proximal 3' splice site for BPV-1 RNA splicing in DT40-ASF cells, a genetically engineered chicken B-cell line that expresses only human ASF/SF2 controlled by a tetracycline-repressible promoter. Depletion of ASF/SF2 from the cells by tetracycline greatly decreased viral RNA expression and RNA splicing at the proximal 3' splice site while increasing use of the distal 3' splice site in the remaining viral RNAs. Activation of cells lacking ASF/SF2 through anti-immunoglobulin M-B-cell receptor cross-linking rescued viral RNA expression and splicing at the proximal 3' splice site and enhanced Akt phosphorylation and expression of the phosphorylated serine/arginine-rich (SR) proteins SRp30s (especially SC35) and SRp40. Treatment with wortmannin, a specific phosphatidylinositol 3-kinase/Akt kinase inhibitor, completely blocked the activation-induced activities. ASF/SF2 thus plays an important role in viral RNA expression and splicing at the proximal 3' splice site, but activation-rescued viral RNA expression and splicing in ASF/SF2-depleted cells is mediated through the phosphatidylinositol 3-kinase/Akt pathway and is associated with the enhanced expression of other SR proteins.     

Novel exploitation of a nuclear function by influenza virus: the cellular SF2/ASF splicing factor controls the amount of the essential viral M2 ion channel protein in infected cells.

We show that a cellular nuclear protein, the SR splicing factor SF2/ASF, controls the level of production of an essential influenza virus protein, the M2 ion channel protein. The M2 mRNA that encodes the ion channel protein is produced by alternative splicing of another viral mRNA, M1 mRNA. The production of M2 mRNA is controlled in two ways. First, a distal (stronger) 5' splice site in M1 mRNA is blocked by the complex of viral polymerase proteins synthesized during infection, allowing the cellular splicing machinery to switch to the proximal (weaker) M2 5' splice site. Second, utilization of the weak M2 5' splice site requires its activation by the cellular SF2/ASF protein. This activation is mediated by the binding of the SF2/ASF protein to a purine-rich splicing enhancer sequence that is located in the 3' exon of M1 mRNA. We demonstrate that activation of the M2 5' splice site is controlled by the SF2/ASF protein in vivo during influenza virus infection. Utilizing four cell lines that differ in their levels of production of the SF2/ASF protein, we show that during virus infection of these cell lines both M2 mRNA and the M2 ion channel protein are produced in amounts that are proportional to the different expression levels of the SF2/ASF protein.     

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.Human papillomavirus type 16 (HPV-16) is the foremost cause of cervical cancer, which is one of the most common cancers in women globally (10, 37). Persistence of high-risk HPV types, such as HPV-16, is the highest risk factor for the development of cervical cancer. The majority of all DNA viruses that establish persistence have evolved a highly organized gene expression program, often divided into clear early and late phases. The HPV-16 genome contains an early promoter that could potentially express mRNAs encoding all viral gene products, and a late differentiation-dependent promoter that specifically excludes expression of E6 and E7 (21). The switch from early to late gene expression includes a promoter switch as well as derepression and activation of the late poly(A) signal and late splice sites (16). To activate late splice sites and the late poly(A) signal, many early splice sites and the early poly(A) signal must be downregulated to allow for competition from mutually exclusive late splice sites and poly(A) signal (8, 26, 36). Other HPV-16 splice sites are used by both early and late mRNAs and should function well in both mitotic cells and terminally differentiated cells. One of the major splice sites used by both early and late mRNAs is SA3358 (Fig. ​(Fig.1A).1A). This splice site is outstanding in that it is used to produce the majority of all HPV-16 mRNAs, including the mRNAs of the oncogenes E6 and E7 and the E4, E5, L1, and perhaps L2 proteins. In contrast, efficient usage of SA3358 specifically prevents expression of HPV-16 E1 and E2.Open in a separate windowFIG. 1.(A) Schematic representation of the HPV-16 genome. Early and late viral promoters p97 and p670 are indicated. Numbers indicate nucleotide positions of 5′-splice sites (filled circles), 3′-splice sites (open circles), or early and late poly(A) signals pAE and pAL, respectively. LCR, long control region. A few selected early and late mRNAs are shown (1). Previously described splicing silencers and enhancers are indicated (24, 34, 35). (B) Diagram with potential ASF/SF2 sites upstream and downstream of SD3632 predicted by ESEfinder (4). Heights of the bars represent degrees of similarity to ASF/SF2 binding sites according to ESEfinder. HPV-16 splice sites SA3358 and SD3632 are indicated. Numbers indicate nucleotide positions in the HPV-16 genome. The position of a previously described enhancer is indicated (24). (C) ASF/SF2 sites in the mutant HPV-16 sequence in which the ASF/SF2 sites had been inactivated, as predicted by ESEfinder (4). (D) Exact sequences of the wt and mutant (mut) HPV-16 Predicted sequences between nucleotide positions 3407 and 3627 in the HPV-16 genome. Dots represent identical nucleotides.Many, if not all, HPV types contain a 3′-splice site in the E4 open reading frame (orf) that is spliced to an upstream 5′-splice site that joins the E1 AUG with the E4 orf. In HPV-16, these splice sites are named SA3358 and SD880 (Fig. ​(Fig.1A),1A), whereas they are named SD847 and SA3325 in HPV-11 and SD877 and SA3295 in HPV-31 (1). Splicing between HPV-16 SD880 and SA3358 (6, 9, 27), or the corresponding sites in HPV-11 (5, 20, 23) and HPV-31 (11, 12), occurs on the most-common early mRNAs encoding E6 and E7, as well as on the most-abundant late mRNA encoding E4. In addition, the most-common L1 mRNA is also spliced between SD880 and SA3358 (17), or the corresponding sites in HPV-11 (23) and HPV-31 (12, 22). Analysis of HPV-16 splicing in cervical scrape samples revealed that splicing between SD880 and SA3358 was the most-common splicing event in both low- and high-grade cervical lesions (25). In vitro transfection experiments demonstrated that splicing to SA3358 was required for efficient expression of E6 and E7 (2). As a matter of fact, splicing between SD880 and SA3358 was required for production of E6 and E7 quantities that were needed for transformation of cells by these HPV proteins. In HPV-31, SA3295 corresponds to HPV-16 SA3358. Mutational inactivation of HPV-31 SA3295 in an infectious molecular clone of HPV-31 immediately caused splicing to a cryptic 3′-splice site located three nucleotides further down (15). These results indicated that HPV-31 SA3295 is under the control of strong splicing enhancer elements and that there is a strong pressure on the virus to maintain a 3′-splice site in that exact region.We have previously reported that HPV-16 SA3358 has an exceptionally poor 3′-splice site sequence compared to a consensus 3′-splice site (24). This is due primarily to an almost complete absence of an upstream row of uninterrupted pyrimidines that normally characterize an efficiently utilized 3′-splice site. However, SA3358 is one of the most efficiently used splice sites on the HPV-16 genome (24, 33). We have previously shown that utilization of HPV-16 SA3358 is totally dependent on exonic sequences downstream of SA3358, and we concluded that a splicing enhancer was located downstream of SA3358 (24). Here, we have followed up these findings; we demonstrate that the enhancer elements downstream of HPV-16 SA3358 are binding sites for ASF/SF2, and we show that ASF/SF2 enhances splicing to SA3358.     

Alternative splicing factor or splicing factor-2 plays a key role in intron retention of the endoglin gene during endothelial senescence

Alternative splicing involving intron retention plays a key role in the regulation of gene expression. We previously reported that the alternatively spliced short isoform of endoglin (S-endoglin) is induced during the aging or senescence of endothelial cells by a mechanism of intron retention. In this work, we demonstrate that the alternative splicing factor or splicing factor-2 (ASF/SF2) is involved in the synthesis of endoglin. Overexpression of ASF/SF2 in endothelial cells switched the balance between the two endoglin isoforms, favoring the synthesis of S-endoglin. Using a minigene reporter vector and RNA immunoprecipitation experiments, it was shown that ASF/SF2 interacts with the nucleotide sequence of the endoglin minigene, suggesting the direct involvement of ASF/SF2. Accordingly, the sequence recognized by ASF/SF2 in the endoglin gene was identified inside the retained intron near the consensus branch point. Finally, the ASF/SF2 subcellular localization during endothelial senescence showed a preferential scattered distribution throughout the cytoplasm, where it interferes with the activity of the minor spliceosome, leading to an increased expression of S-endoglin mRNA. In summary, we report for the first time the molecular mechanisms by which ASF/SF2 regulates the alternative splicing of endoglin in senescent endothelial cells, as well as the involvement of ASF/SF2 in the minor spliceosome.