Inhibition of the Akt kinase activates HPV16 late gene expression by reducing HPV16 early polyadenylation and by activating HPV16 late L1 mRNA splicing. We identified ‘hot spots’ for RNA binding proteins at the early polyA signal and at splice sites on HPV16 late mRNAs. We observed that hnRNP L was associated with sequences at all HPV16 late splice sites and at the early polyA signal. Akt kinase inhibition resulted in hnRNP L dephosphorylation and reduced association of hnRNP L with HPV16 mRNAs. This was accompanied by an increased binding of U2AF65 and Sam68 to HPV16 mRNAs. Furthermore, siRNA knock-down of hnRNP L or Akt induced HPV16 gene expression. Treatment of HPV16 immortalized keratinocytes with Akt kinase inhibitor reduced hnRNP L binding to HPV16 mRNAs and induced HPV16 L1 mRNA production. Finally, deletion of the hnRNP L binding sites in HPV16 subgenomic expression plasmids resulted in activation of HPV16 late gene expression. In conclusion, the Akt kinase inhibits HPV16 late gene expression at the level of RNA processing by controlling the RNA-binding protein hnRNP L. We speculate that Akt kinase activity upholds an intracellular milieu that favours HPV16 early gene expression and suppresses HPV16 late gene expression.

We show that the alkylating cancer drug melphalan activated the DNA damage response and induced human papillomavirus type 16 (HPV16) late gene expression in an ATM- and Chk1/2-dependent manner. Activation of HPV16 late gene expression included inhibition of the HPV16 early polyadenylation signal that resulted in read-through into the late region of HPV16. This was followed by activation of the exclusively late, HPV16 splice sites SD3632 and SA5639 and production of spliced late L1 mRNAs. Altered HPV16 mRNA processing was paralleled by increased association of phosphorylated BRCA1, BARD1, BCLAF1 and TRAP150 with HPV16 DNA, and increased association of RNA processing factors U2AF65 and hnRNP C with HPV16 mRNAs. These RNA processing factors inhibited HPV16 early polyadenylation and enhanced HPV16 late mRNA splicing, thereby activating HPV16 late gene expression.

We report that many histone modifications are unevenly distributed over the HPV16 genome in cervical cancer cells as well as in HPV16-immortalized keratinocytes. For example, H3K36me3 and H3K9Ac that are common in highly expressed cellular genes and over exons, were more common in the early than in the late region of the HPV16 genome. In contrast, H3K9me3, H4K20me3, H2BK5me1 and H4K16Ac were more frequent in the HPV16 late region. Furthermore, a region encompassing the HPV16 early polyadenylation signal pAE displayed high levels of histone H3 acetylation. Histone deacetylase (HDAC) inhibitors caused a 2- to 8-fold induction of HPV16 early and late mRNAs in cervical cancer cells and in immortalized keratinocytes, while at the same time increasing the levels of acetylated histones in the cells and on the HPV16 genome specifically. We concluded that increased histone acetylation on the HPV16 genome correlates with increased HPV16 gene expression.

Human papillomavirus type 16 (HPV16) E2 is an essential HPV16 protein. We have investigated how HPV16 E2 expression is regulated and have identifed a splicing enhancer that is required for production of HPV16 E2 mRNAs. This uridine-less splicing enhancer sequence (ACGAGGACGAGGACAAGGA) contains 84% adenosine and guanosine and 16% cytosine and consists of three ‘AC(A/G)AGG’-repeats. Mutational inactivation of the splicing enhancer reduced splicing to E2-mRNA specific splice site SA2709 and resulted in increased levels of unspliced E1-encoding mRNAs. The splicing enhancer sequence interacted with cellular RNA binding protein hnRNP G that promoted splicing to SA2709 and enhanced E2 mRNA production. The splicing-enhancing function of hnRNP G mapped to amino acids 236–286 of hnRNP G that were also shown to interact with splicing factor U2AF65. The interactions between hnRNP G and HPV16 E2 mRNAs and U2AF65 increased in response to keratinocyte differentiation as well as by the induction of the DNA damage response (DDR). The DDR reduced sumoylation of hnRNP G and pharmacological inhibition of sumoylation enhanced HPV16 E2 mRNA splicing and interactions between hnRNP G and E2 mRNAs and U2AF65. Intriguingly, hnRNP G also promoted intron retention of the HPV16 E6 coding region thereby inhibiting production of spliced E7 oncogene mRNAs.

The Chinese forest musk deer ( Moschus berezovskii ; FMD) is an artiodactyl mammal and is both economically valuable and highly endangered. To investigate the genetic mechanisms of musk secretion and adaptive immunity in FMD, we compared its genome to nine other artiodactyl genomes. Comparative genomics demonstrated that eight positively selected genes (PSGs) in FMD were annotated in three KEGG pathways that were related to metabolic and synthetic activity of musk, similar to previous transcriptome studies. Functional enrichment analysis indicated that many PSGs were involved in the regulation of immune system processes, implying important reorganization of the immune system in FMD. FMD-specific missense mutations were found in two PSGs ( MHC class II antigen DRA and ADA ) that were classified as deleterious by PolyPhen-2, possibly contributing to immune adaptation to infectious diseases. Functional assessment showed that the FMD-specific mutation enhanced the ADA activity, which was likely to strengthen the immune defense against pathogenic invasion. Single nucleotide polymorphism-based inference showed the recent demographic trajectory for FMD. Our data and findings provide valuable genomic resources not only for studying the genetic mechanisms of musk secretion and adaptive immunity, but also for facilitating more effective management of the captive breeding programs for this endangered species.

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The purinergic signaling has an important role in regulating pancreatic exocrine secretion. The exocrine pancreas is also a site of one of the most serious cancer forms, the pancreatic ductal adenocarcinoma (PDAC). Here, we explore how the network of purinergic and adenosine receptors, as well as ecto-nucleotidases regulate normal pancreatic cells and various cells within the pancreatic tumor microenvironment. In particular, we focus on the P2X7 receptor, P2Y2 and P2Y12 receptors, as well as A2 receptors and ecto-nucleotidases CD39 and CD73. Recent studies indicate that targeting one or more of these candidates could present new therapeutic approaches to treat pancreatic cancer. In pancreatic cancer, as much as possible of normal pancreatic function should be preserved, and therefore physiology of purinergic signaling in pancreas needs to be considered.

Here we show that treatment of the HPV16‐positive tonsillar cancer cell line HN26 with DNA alkylating cancer drug melphalan‐induced p53 and activated apoptosis. Melphalan reduced the levels of RNA polymerase II and cellular transcription factor Sp1 that were associated with HPV16 DNA. The resulting inhibition of transcription caused a rapid loss of the HPV16 early mRNAs encoding E6 and E7 as a result of their inherent instability. As a consequence of HPV16 E6 and E7 down‐regulation, the DNA damage inflicted on the cells by melphalan caused induction of p53 and activation of apoptosis in the HN26 cells. The BARD1‐negative phenotype of the HN26 cells may have contributed to the failure to repair DNA damage caused by melphalan, as well as to the efficient apoptosis induction. Finally, nude mice carrying the HPV16 positive tonsillar cancer cells responded better to melphalan than to cisplatin, the chemotherapeutic drug of choice for tonsillar cancer. We concluded that the short half‐life of the HPV16 E6 and E7 mRNAs renders HPV16‐driven tonsillar cancer cells particularly sensitive to DNA damaging agents such as melphalan since melphalan both inhibits transcription and causes DNA damage.