We have previously identified cis-acting RNA sequences in the human papillomavirus type 16 (HPV-16) L1 coding region which inhibit expression of L1 from eukaryotic expression plasmids. Here we have determined the function of one of these RNA elements, and we provide evidence that this RNA element is a splicing silencer which suppresses the use of the 3 splice site located immediately upstream of the L1 AUG. We also show that this splice site is inefficiently utilized as a result of a suboptimal polypyrimidine tract. Introduction of point mutations in the L1 coding region that altered the RNA sequence without affecting the L1 protein sequence resulted in the inactivation of the splicing silencer and induced splicing to the L1 3 splice site. These mutations also prevented the interaction of the RNA silencer with a 35-kDa cellular protein identified here as hnRNP A1. The splicing silencer in L1 inhibits splicing in vitro, and splicing can be restored by the addition of RNAs containing an hnRNP A1 binding site to the reaction, demonstrating that hnRNP A1 inhibits splicing of the late HPV-16 mRNAs through the splicing silencer sequence. While we show that one role of the splicing silencer is to determine the ratio between partially spliced L2/L1 mRNAs and spliced L1 mRNAs, we also demonstrate that it inhibits splicing from the major 5 splice site in the early region to the L1 3 splice site, thereby playing an essential role in preventing late gene expression at an early stage of the viral life cycle. We speculate that the activity of the splicing silencer and possibly the concentration of hnRNP A1 in the HPV-16-infected cell determines the ability of the virus to establish a persistent infection which remains undetected by the host immune surveillance.Human papillomaviruses (HPVs) are a group of nonenveloped, double-stranded DNA tumor viruses with tropism for epithelial cells (21, 62). HPV type 16 (HPV-16) is one of the most common sexually transmitted HPV types and is also the HPV type most frequently detected in cervical cancers (43). While many of the early HPV gene products are present in all layers of the squamous epithelium, expression of the late mRNAs encoding the L1 and L2 capsid proteins is restricted to the terminally differentiated cells in the upper layers of the epithelium (21). For this reason, it has been difficult to grow HPVs in vitro, and only organotypic raft cultures in which infected or transfected keratinocytes are grown in the airliquid interface to induce terminal cell differentiation produce HPV virions (27). Expression of the late genes is regulated at the levels of transcription and RNA processing (1,38,39,42,55). Members of our group and others have previously shown that the late HPV-16 mRNAs contain cis-acting regulatory RNA elements (23, 24, 51), both in the 3Ј untranslated region (UTR) and in the L1 and L2 coding regions (9,33,46,51). It is likely that these elements are involved in the regulation of HPV late gene expression. Inhibitory RNA elements in the late 3Ј UTR have been identif...

We have investigated the role of the human papillomavirus type 16 (HPV-16) early untranslated region (3 UTR) in HPV-16 gene expression. We found that deletion of the early 3 UTR reduced the utilization of the early polyadenylation signal and, as a consequence, resulted in read-through into the late region and production of late L1 and L2 mRNAs. Deletion of the U-rich 3 half of the early 3 UTR had a similar effect, demonstrating that the 57-nucleotide U-rich region acted as an enhancing upstream element on the early polyadenylation signal. In accordance with this, the newly identified hFip1 protein, which has been shown to enhance polyadenylation through U-rich upstream elements, interacted specifically with the HPV-16 upstream element. This upstream element also interacted specifically with CstF-64, hnRNP C1/C2, and polypyrimidine tract binding protein, suggesting that these factors were either enhancing or regulating polyadenylation at the HPV-16 early polyadenylation signal. Mutational inactivation of the early polyadenylation signal also resulted in increased late mRNA production. However, the effect was reduced by the activation of upstream cryptic polyadenylation signals, demonstrating the presence of additional strong RNA elements downstream of the early polyadenylation signal that direct cleavage and polyadenylation to this region of the HPV-16 genome. In addition, we identified a 3 splice site at genomic position 742 in the early region with the potential to produce E1 and E4 mRNAs on which the E1 and E4 open reading frames are preceded only by the suboptimal E6 AUG. These mRNAs would therefore be more efficiently translated into E1 and E4 than previously described HPV-16 E1 and E4 mRNAs on which E1 and E4 are preceded by both E6 and E7 AUGs.Human papillomaviruses (HPVs) are a group of nonenveloped, double-stranded DNA tumor viruses with tropism for epithelial cells (21). HPV-16 is one of the most common sexually transmitted HPV types and is also the HPV type most frequently detected in cervical cancer (42,63). The life cycle of HPV-16 is strictly linked to the differentiation program of the infected cell (17,21). While many of the early HPV gene products are present in all layers of the squamous epithelium, expression of the late mRNAs encoding the L1 and L2 capsid proteins is restricted to the terminally differentiated cells in the upper layers of the epithelium (29). The early and late regions are separated by the early polyadenylation signal named pAE (Fig. 1), and RNA elements that regulate the use of the pAE signal are likely to affect late gene expression. Here we have studied the role of the early untranslated region (3Ј UTR) in late gene expression by investigating how deletions in the early 3Ј UTR affect late mRNA levels.The 3Ј UTR is often the site for RNA elements that regulate various steps in the mRNA processing pathway, for example, mRNA transport, half-life, and translation (13). The best-studied 3Ј-UTR element is the group of AU-rich RNA instability elements, often containing multiple co...

Successful inhibition of human papillomavirus type 16 (HPV-16) late gene expression early in the life cycle is essential for persistence of infection, the highest risk factor for cervical cancer. Our study aimed to locate regulatory RNA elements in the early region of HPV-16 that influence late gene expression. For this purpose, subgenomic HPV-16 expression plasmids under control of the strong human cytomegalovirus immediate early promoter were used. An exonic splicing enhancer that firmly supported the use of the E4 3 splice site at position 3358 in the early region of the HPV-16 genome was identified. The enhancer was mapped to a 65-nucleotide AC-rich sequence located approximately 100 nucleotides downstream of the position 3358 3 splice site. Deletion of the enhancer caused loss of both splicing at the upstream position 3358 3 splice site and polyadenylation at the early polyadenylation signal, pAE. Direct splicing occurred at the competing L1 3 splice site at position 5639 in the late region. Optimization of the position 3358 3 splice site restored splicing to that site and polyadenylation at pAE. Additionally, a sequence of 40 nucleotides with a negative effect on late mRNA production was located immediately downstream of the enhancer. As the E4 3 splice site is employed by both early and late mRNAs, the enhancer constitutes a key regulator of temporal HPV-16 gene expression, which is required for early mRNA production as well as for the inhibition of premature late gene expression.Human papillomavirus type 16 (HPV-16) is the major cause of cervical cancer (41, 60) and is one of the most common cancer-causing infections in the world. Cervical cancer accounts for 6% of all malignancies in women in developed countries but 26% in developing countries (37). While the majority of HPV-16 infections are cleared in less than a year, in rare cases, despite immunosurveillance, persistence is established. Persistence of HPV-16 infection is itself the highest risk factor for the development of cervical cancer (28) and requires successful inhibition of late gene expression early in the life cycle to avoid the immune response. As cervical cancer cells never express the late viral mRNAs or proteins, one can speculate that inhibition of late gene expression is a prerequisite for cancer progression.Expression of HPV-16 genes is complex and is tightly regulated by differentiation-dependent transcription and RNA processing events (2, 40). Production of the late mRNAs encoding the antigenic capsid proteins L1 and L2 is restricted to terminally differentiated keratinocytes (41). The inhibition of the late genes may be partly attributed to RNA elements in the late region, suggested to regulate various RNA processing events such as mRNA stability, splicing, and translation (2, 40). Regulatory RNA elements have been identified in HPV-16 coding regions (42,45) and also in the late untranslated region of HPV-1 (46), HPV-16 (31), HPV-31 (13), and bovine papillomavirus type 1 (BPV-1) (23), while differentiation-dependent splicing of t...

Polycomb-mediated gene silencing and DNA methylation underlie many epigenetic processes important in normal development as well as in cancer. An interaction between EZH2 of the Polycomb repressive complex 2 (PRC2), which trimethylates lysine 27 on Histone 3 (H3K27me3), and all three DNA methyltransferases (DNMTs) has been reported, implicating a role for PRC2 in directing DNA methylation. Interestingly, however, the majority of H3K27me3 marked genes lack DNA methylation in ES cells, indicating that EZH2 recruitment may not be sufficient to promote DNA methylation. Here, we employed a Gal4DBD/gal4UAS-based system to directly test if EZH2 binding at a defined genomic site is sufficient to promote de novo DNA methylation in a murine erythroleukaemia cell line. Targeting of a Gal4DBD-EZH2 fusion to an intergenic transgene bearing a gal4 binding-site array promoted localized recruitment of Suz12 and Bmi1, subunits of PRC2 and PRC1, respectively, and deposition of H3K27me3. Further analysis of the H3K27me3-marked site revealed the persistence of H3K4me2, a mark inversely correlated with DNA methylation. Strikingly, while Dnmt3a was also recruited in an EZH2-dependent manner, de novo DNA methylation of the transgene was not observed. Thus, while targeting of EZH2 to a specific genomic site is sufficient for recruitment of Dnmt3a, additional events are required for de novo DNA methylation.

The human papillomavirus type 1 (HPV-1) late mRNAs contain a 57-nucleotide adenosine-and uridine-rich RNA instability element termed h1ARE in their late 3 untranslated regions. Here we show that five sequence motifs in the h1ARE (named I-V) affect the mRNA halflife in an additive manner. The minimal inhibitory sequence in motifs I and II was mapped to UAUUUAU, and the minimal inhibitory sequence in motifs III-V was mapped to UAUUUUUAU. We also provide evidence that the same motifs in the AU-RNA instability element inhibit mRNA translation, an effect that was entirely dependent on the presence of a poly(A) tail on the mRNA. Additional experiments demonstrated that the h1ARE interacted directly with the poly(A)-binding protein, suggesting that the h1ARE inhibits translation by interfering with the function of the poly(A)-binding protein.Human papillomaviruses (HPVs) 1 are a group of non-enveloped, double-stranded DNA tumor viruses with tropism for epithelial cells (1, 2). Expression of the late mRNAs is restricted to the terminally differentiated cells in the upper layers of the epithelium and at least four papillomaviruses (bovine papillomavirus type 1 (BPV-1), HPV-1, -16, and -31) have been shown to contain cis-acting inhibitory RNA elements located in the late 3Ј UTR (reviewed in Refs. 3-6). In addition, negative RNA elements have been identified in the HPV-16 L1 and L2 open reading frames (4,7,8).We have previously identified and characterized an inhibitory AU-rich element (ARE) located in the HPV-1 late 3Ј UTR region named h1ARE (4 -6, 9) ( Fig. 1). Using actinomycin D we showed that the presence of the h1ARE reduced the mRNA half-life (10). The minimal inhibitory sequence termed XB spans 57 nucleotides (nt) and contains 93% A and U. The element contains two AUUUA-and the three UUUUU-containing sequences (9, 10). Replacing two uridines (U) with cytidines (C) in each motif inactivated the h1ARE (10). The h1ARE interacts with cellular factors (11,12), which bind to the c-fos ARE (10). Two of the h1ARE binding factors interacted with the wild type h1ARE but not with a functionally inactive mutant of the h1ARE (10). These proteins were identified as HuR and hnRNP C (10, 13), and we later showed that binding of the HuR protein correlates with inhibitory activity of a panel of h1ARE mutants (13). While HuR binds to both AUUUA-and UUUUU-motifs (13), hnRNP C binds exclusively to the UUUUU-motifs (14). The role of hnRNP C in HPV-1 late gene expression is unclear. The HuR protein shuttles between the nucleus and the cytoplasm (15), and we observed that there was an inverse correlation between the levels of HuR in the cell cytoplasm and the inhibitory activity of the h1ARE (16), suggesting that the presence of high levels of HuR in the cytoplasm antagonizes the inhibitory effect of the h1ARE, whereas a primarily nuclear association of HuR is associated with inhibition of HPV-1 late gene expression. Interestingly, the HIV Rev and RRE, and the SRV-1 CTE can overcome the inhibition (9), suggesting that the h1ARE traps th...

Malaria infects 500 million people annually, a number that is likely to rise as drug resistance to currently used antimalarials increases. During its intraerythrocytic stage, the causative parasite, Plasmodium falciparum, metabolizes hemoglobin and releases toxic heme, which is neutralized by a parasite-specific crystallization mechanism to form hemozoin. Evidence suggests that chloroquine, the most successful antimalarial agent in history, acts by disrupting the formation of hemozoin. Here we describe the development of a 384-well microtiter plate screen to detect small molecules that can also disrupt heme crystallization. This assay, which is based on a colorimetric assay developed by Ncokazi and Egan (K. K. Ncokazi and T. J. Egan, Anal. Biochem. 338:306-319, 2005), requires no parasites or parasite-derived reagents and no radioactive materials and is suitable for a high-throughput screening platform. The assay's reproducibility and large dynamic range are reflected by a Z factor of 0.74. A pilot screen of 16,000 small molecules belonging to diverse structural classes was conducted. The results of the target-based assay were compared with a whole-parasite viability assay of the same small molecules to identify small molecules active in both assays.

Two splice sites on the human papillomavirus type 16 (HPV-16) genome are used exclusively by the late capsid protein L1 mRNAs: SD3632 and SA5639. These splice sites are suppressed in mitotic cells. This study showed that serine/arginine-rich protein 30c (SRp30c), also named SFRS9, activated both SD3632 and SA5639 and induced production of L1 mRNA. Activation of HPV-16 L1 mRNA splicing by SRp30c required an intact arginine/serine-repeat (RS) domain of SRp30c. In addition to this effect, SRp30c could enhance L1 mRNA production indirectly by inhibiting the early 39-splice site SA3358, which competed with the late 39-splice site SA5639. SRp30c bound directly to sequences downstream of SA3358, suggesting that SRp30c inhibited the enhancer at SA3358 and caused a redirection of splicing to the late 39-splice site SA5639. This inhibitory effect of SRp30c was independent of its RS domain. These results suggest that SRp30c can activate HPV-16 L1 mRNA expression via a bimodal mechanism: directly by stimulating splicing to late splice sites and indirectly by inhibiting competing early splice sites.

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