Single-stranded circular RNAs (circRNAs), generated through ‘backsplicing’, occur more extensively than initially anticipated. The possible functions of the vast majority of circRNAs remain unknown. Virus-derived circRNAs have recently been described in gamma-herpesviruses. We report that oncogenic human papillomaviruses (HPVs) generate circRNAs, some of which encompass the E7 oncogene (circE7). HPV16 circE7 is detectable by both inverse RT-PCR and northern blotting of HPV16-transformed cells. CircE7 is N 6 -methyladenosine (m 6 A) modified, preferentially localized to the cytoplasm, associated with polysomes, and translated to produce E7 oncoprotein. Specific disruption of circE7 in CaSki cervical carcinoma cells reduces E7 protein levels and inhibits cancer cell growth both in vitro and in tumor xenografts. CircE7 is present in TCGA RNA-Seq data from HPV-positive cancers and in cell lines with only episomal HPVs. These results provide evidence that virus-derived, protein-encoding circular RNAs are biologically functional and linked to the transforming properties of some HPV.

Proliferating cells depend on glucose uptake more than quiescent cells for their growth. While glucose transport in keratinocytes is mediated largely by the Glut1 facilitative transporter, the keratinocyte-specific ablation of Glut1 did not compromise mouse skin development and homeostasis. Ex vivo metabolic profiling revealed altered sphingolipid, hexose, amino acid, and nucleotide metabolism in Glut1-deficient keratinocytes, suggesting metabolic adaptation. On the other hand, Glut1-deficient keratinocytes in culture displayed metabolic and oxidative stress and impaired proliferation. Similarly, Glut1 deficiency impaired in vivo keratinocyte proliferation and migration within wounded or UV-damaged mouse skin. Notably, both genetic and pharmacological Glut1 inactivation reduced hyperplasia in mouse models of psoriasis-like disease. Topical application of a Glut1 inhibitor also reduced inflammation in these models. Glut1 inhibition decreased expression of pathology-associated genes in human psoriatic skin organoids. Thus, Glut1 is selectively required for injury- and inflammation-associated keratinocyte proliferation, and its inhibition offers a novel treatment strategy for psoriasis.

Mammalian biological processes such as inflammation, involve regulation of hundreds of genes controlling onset and termination. MicroRNAs (miRNAs) can translationally repress target mRNAs and regulate innate immune responses. Our model system comprised primary human keratinocytes, which exhibited robust differences in inflammatory cytokine production (interleukin-6 and tumor necrosis factor-␣) following specific Toll-like receptor 2 and 4 (TLR-2/TLR-4) agonist challenge. We challenged these primary cells with Porphyromonas gingivalis (a Gram-negative bacterium that triggers TLR-2 and TLR-4) and performed miRNA expression profiling. We identified miRNA (miR)-105 as a modulator of TLR-2 protein translation in human gingival keratinocytes. There was a strong inverse correlation between cells that had high cytokine responses following TLR-2 agonist challenge and miR-105 levels. Knock-in and knock-down of miR-105 confirmed this inverse relationship. In silico analysis predicted that miR-105 had complementarity for TLR-2 mRNA, and the luciferase reporter assay verified this. Further understanding of the role of miRNA in host responses may elucidate disease susceptibility and suggest new anti-inflammatory therapeutics.

RBM10 is an RNA splicing regulator that is frequently mutated in lung adenocarcinoma (LUAD) and has recently been proposed to be a cancer gene. How RBM10 mutations observed in LUAD affect its normal functions, however, remains largely unknown. Here integrative analysis of RBM10 mutation and RNA expression data revealed that LUAD-associated RBM10 mutations exhibit a mutational spectrum similar to that of tumor suppressor genes. In addition, this analysis showed that RBM10 mutations identified in LUAD patients lacking canonical oncogenes are associated with significantly reduced RBM10 expression. To systematically investigate RBM10 mutations, we developed an experimental pipeline for elucidating their functional effects. Among six representative LUAD-associated RBM10 mutations, one nonsense and one frameshift mutation caused loss-of-function as expected, whereas four missense mutations differentially affected RBM10-mediated splicing. Importantly, changes in proliferation rates of LUAD-derived cells caused by these RBM10 missense mutants correlated with alterations in RNA splicing of RBM10 target genes. Together, our data implies that RBM10 mutations contribute to LUAD pathogenesis, at least in large part, by deregulating splicing. The methods described in this study should be useful for analyzing mutations in additional cancer-associated RNA splicing regulators.

Mutations in the spliceosomal RNA binding protein RBM10 cause TARP syndrome and are frequently observed in lung adenocarcinoma (LUAD). We have previously shown that RBM10 enhances exon skipping of its target genes, including its paralog RBM5. Here, we report that RBM10 negatively regulates its own mRNA and protein expression and that of RBM5 by promoting alternative splicing-coupled nonsense-mediated mRNA decay (AS-NMD). Through computational analysis and experimental validation, we identified RBM10-promoted skipping of exon 6 or 12 in RBM10 and exon 6 or 16 in RBM5 as the underlying AS-NMD events. Importantly, we showed that LUAD-associated mutations affecting splice sites of RBM10 exons 6 or 12 abolished exon inclusion and correlated with reduced expression of RBM10 RNA. Together, our investigations have revealed novel molecular mechanisms underlying RBM10 autoregulation and cross-regulation of RBM5, thereby providing insights concerning the functions of RBM10 under various physiological and pathological conditions. Our combined computational and experimental approach should be useful for elucidating the role of AS-NMD in auto- and cross-regulation by other splicing regulators.

Periodontitis is a chronic human inflammatory disease initiated and sustained by dental plaque microorganisms. A major contributing pathogen is Porphyromonas gingivalis, a gram-negative bacterium recognized by Toll-like receptor 2 (TLR2) and TLR4, which are expressed by human gingival epithelial cells (HGECs). However, it is still unclear how these cells respond to P. gingivalis and initiate inflammatory and immune responses. We have reported previously that HGECs produce a wide range of proinflammatory cytokines, including interleukin-6 (IL-6), IL-8, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor alpha (TNF-␣), and IL-1␤. In this study, we show that IL-1␤ has a special role in the modulation of other inflammatory cytokines in HGECs challenged with P. gingivalis. Our results show that the increased production of IL-1␤ correlates with the cell surface expression of TLR4, and more specifically, TLR4-normal HGECs produce fourfold more IL-1␤ than do TLR4-deficient HGECs after challenge. Moreover, blocking the IL-1␤ receptor greatly reduces the production of "secondary" proinflammatory cytokines such as IL-8 or IL-6. Our data indicate that the induction of IL-1␤ plays an important role in mediating the release of other proinflammatory cytokines from primary human epithelial cells following challenge with P. gingivalis, and this process may be an inflammatory enhancement mechanism adopted by epithelial cells.

Recent studies implicate the mammalian target of rapamycin (mTOR) pathway in the control of inflammatory responses following Toll-like receptor (TLR) stimulation in myeloid cells but its role in non-myeloid cells such as human keratinocytes is unknown. Here we show that TLR3 signaling can induce robust cytokine secretion including interleukin 1 beta (IL-1β), tumor necrosis factor alpha (TNFα), IL-12p70 and interferon beta (IFN-β), and our data reveal for the first time that inhibiting mTOR with rapamycin, suppresses these TLR3 induced responses but actually enhances bioactive IL-12p70 production in human oral keratinocytes. Rapamycin inhibited the phosphorylation of the 70-kDa ribosomal protein S6 kinase (p70S6K) and the 4E binding protein 1 (4EBP-1), and suppressed the mitogen activated protein kinase (MAPK) pathway by decreasing phosphorylation of c-Jun N-terminal kinase (JNK). We also show that TLR3 induces interferon regulatory factor 3 (IRF3) activation by Akt via an mTOR-p70S6K-4EBP1 pathway. Furthermore, we provide evidence that Poly I:C induced expression of IL-1β, TNFα, IL-12p70 and IFN-β was blocked by JNK inhibitor SP600125. TLR3 preferentially phosphorylated IKKα through mTOR to activate nuclear factor kappa beta (NF-kB) in human oral keratinocytes. Taken together, these data demonstrate p70S6K, p4EBP1, JNK, NF-kB and IRF3 are involved in the regulation of inflammatory mediators by TLR3 via the mTOR pathway. mTOR is a novel pathway modulating TLR3 induced inflammatory and antiviral responses in human oral keratinocytes.

SummaryPorphyromonas gingivalis, a Gram-negative oral pathogen, has been shown to induce apoptosis in human gingival epithelial cells, yet the underlining cellular mechanisms controlling this process are poorly understood. We have previously shown that the P. gingivalis proteases arginine and lysine gingipains, are necessary and sufficient to induce host cell apoptosis. In the present study, we demonstrate that 'P. gingivalis-induced apoptosis' is mediated through degradation of actin leading to cytoskeleton collapse. Stimulation of human gingival epithelial cells with P. gingivalis strains 33277 and W50 at moi:100 induced b-actin cleavage as early as 1 h and human serum inhibited this effect. By using gingipain-deficient mutants of P. gingivalis and purified gingipains, we demonstrate that lysine gingipain is involved in actin hydrolysis in a dose and time-dependent manner. Use of Jasplakinolide and cytochalasin D revealed that P. gingivalis internalization is necessary for actin cleavage. Further, we also show that lysine gingipain from P. gingivalis can cleave active caspase 3. Taken together, we have identified actin as a substrate for lysine gingipain and demonstrated a novel mechanism involved in microbial host cell invasion and apoptosis.