SUMMARY Alternative splicing of the PKM2 gene produces two isoforms, M1 and M2, which are preferentially expressed in adult and embryonic tissues, respectively. The M2 isoform is reexpressed in human cancer and has nonmetabolic functions in the nucleus as a protein kinase. Here, we report that PKM2 is acetylated by p300 acetyltransferase at K433, which is unique to PKM2 and directly contacts its allosteric activator, fructose 1,6-bisphosphate (FBP). Acetylation prevents PKM2 activation by interfering with FBP binding and promotes the nuclear accumulation and protein kinase activity of PKM2. Acetylationmimetic PKM2(K433) mutant promotes cell proliferation and tumorigenesis. K433 acetylation is decreased by serum starvation and cell-cell contact, increased by cell cycle stimulation, epidermal growth factor (EGF), and oncoprotein E7, and enriched in breast cancers. Hence, K433 acetylation links cell proliferation and transformation to the switch of PKM2 from a cytoplasmic metabolite kinase to a nuclear protein kinase.

The disruption of the intestinal epithelial barrier function occurs commonly in various pathologies, but the exact mechanisms responsible are unclear. The H19 long noncoding RNA (lncRNA) regulates the expression of different genes and has been implicated in human genetic disorders and cancer. Here, we report that H19 plays an important role in controlling the intestinal epithelial barrier function by serving as a precursor for microRNA 675 (miR-675). H19 overexpression increased the cellular abundance of miR-675, which in turn destabilized and repressed the translation of mRNAs encoding tight junction protein ZO-1 and adherens junction E-cadherin, resulting in the dysfunction of the epithelial barrier. Increasing the level of the RNA-binding protein HuR in cells overexpressing H19 prevented the stimulation of miR-675 processing from H19, promoted ZO-1 and E-cadherin expression, and restored the epithelial barrier function to a nearly normal level. In contrast, the targeted deletion of HuR in intestinal epithelial cells enhanced miR-675 production in the mucosa and delayed the recovery of the gut barrier function after exposure to mesenteric ischemia/reperfusion. These results indicate that H19 interacts with HuR and regulates the intestinal epithelial barrier function via the H19-encoded miR-675 by altering ZO-1 and E-cadherin expression posttranscriptionally.T he majority of the mammalian genome is transcribed into a vast number of noncoding RNAs, whereas protein-coding transcripts account for only a minority of transcriptional output (1, 2). Long noncoding RNAs (lncRNAs) are defined as transcribed RNAs spanning Ͼ200 nucleotides in length that lack protein-coding capacity and are distinct from well-characterized structural RNAs (rRNAs, tRNAs, snRNAs, and snoRNAs) or small regulatory RNAs (3, 4). lncRNAs arise from intergenic, antisense, or promoter-proximal regions, and they share many features with mRNAs. Both classes of RNA can be transcribed from multiexonic genes and possess a 5=-methyl-guanosine cap and 3=-poly(A) tail (1,3,5). Some lncRNAs are ubiquitous, but others are dynamically expressed in tissue-, differentiation stage-, and cell type-specific patterns (4, 6). lncRNAs have been involved in a variety of cellular functions, physiologic processes, and disease states by modulating gene expression at different levels, including chromatin remodeling, transcriptional and posttranscriptional control, and protein metabolism (1, 3, 4). lncRNAs can serve as repressors or activators of gene transcription, as has been extensively reported (1, 4), but lncRNAs also can regulate mRNA decay and translation, working jointly with microRNAs (miRNAs) and RNA-binding proteins (RBPs) (7-9).Intercellular junction complexes, comprising tight junctions (TJs) and adherens junctions (AJs), fence the paracellular space in simple epithelia, such as those lining the intestine, kidneys, and lung, and form an important barrier against a wide array of noxious substances present in the lumen (10, 11). In the intestine, the disruption...

The recent emergence of the human infection confirmed to be caused by severe fever with thrombocytopenia syndrome virus (SFTSV) in China is of global concern. Safe diagnostic immunoreagents for determination of human and animal seroprevalence in epidemiological investigations are urgently needed. This paper describes the cloning and expression of the nucleocapsid (N) protein of SFTSV. An N-protein-based double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) system was set up to detect the total antibodies in human and animal sera. We reasoned that as the double-antigen sandwich ELISA detected total antibodies with a higher sensitivity than traditional indirect ELISA, it could be used to detect SFTSV-specific antibodies from different animal species. The serum neutralization test was used to validate the performance of this ELISA system. All human and animal sera that tested positive in the neutralization test were also positive in the sandwich ELISA, and there was a high correlation between serum neutralizing titers and ELISA readings. Cross-reactivity was evaluated, and the system was found to be highly specific to SFTSV; all hantavirus-and dengue virus-confirmed patient samples were negative. SFTSV-confirmed human and animal sera from both Anhui and Hubei Provinces in China reacted with N protein in this ELISA, suggesting no major antigenic variation between geographically disparate virus isolates and the suitability of this assay in nationwide application. ELISA results showed that 3.6% of the human serum samples and 47.7% of the animal field serum samples were positive for SFTSV antibodies, indicating that SFTSV has circulated widely in China. This assay, which is simple to operate, poses no biohazard risk, does not require sophisticated equipment, and can be used in disease surveillance programs, particularly in the screening of large numbers of samples from various animal species.

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Cervical cancer (CC) is ranked as the fourth most common cancer that occurs in women universally, which normally causes pain in the lower belly. Plenty of studies have stated that the expression of long non-coding RNAs (lncRNAs) is linked to the cellular development of many kinds of cancers. DSCAM-AS1 has been reported to act as an oncogene in other cancer types and the aim of our study was to uncover the function and regulatory mechanism of DSCAM-AS1 in CC. In this research, our findings presented that DSCAM-AS1 expression was up-regulated in CC cells. DSCAM-AS1 led to the development of CC by enhancing cell proliferation, migration and invasion ability. DSCAM-AS1 was verified to combine with miR-877-5p and down-regulate the expression of miR-877-5p. Results also showed that ATXN7L3 was a downstream target gene of miR-877-5p and it was unfavorably modulated by miR-877-5p. Enhanced expression of ATXN7L3 counterbalanced the DSCAM-AS1 knockdown effect on the progression of CC. This was the first time to analyze the underlying regulatory mechanism of the oncogenic DSCAM-AS1. Our findings clarified that DSCAM-AS1 played as an oncogenic lncRNA by targeting miR-877-5p/ATXN7L3 axis to promote CC progression, which may provide insights into the prevention of CC.

The RNA interference (RNAi) pathway directs an important antiviral immunity mechanism in plants and invertebrates. Recently, we and others have demonstrated that the antiviral RNAi response is also conserved in mammals, at least to five distinct RNA viruses, including Zika virus (ZIKV). ZIKV may preferentially infect neuronal progenitor cells (NPCs) in the developing foetal brain. Ex vivo ZIKV infection induces RNAi-mediated antiviral response in human NPCs, but not in the more differentiated NPCs or somatic cells. However, litter is known about the in vivo property or function of the virus-derived small-interfering RNAs (vsiRNAs) targeting ZIKV. Here we report a surprising observation: different from ex vivo observations, viral small RNAs (vsRNAs) targeting ZIKV were produced in vivo upon infection in both central neuron system (CNS) and muscle tissues. In addition, our findings demonstrate the production of canonical vsiRNAs in murine CNS upon antiviral RNAi activation by Sindbis virus (SINV), suggesting the possibility of antiviral immune strategy applied by mammals in the CNS.

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The interferon-regulated antiviral responses are essential for the induction of both innate and adaptive immunity in mammals. Production of virus-derived small-interfering RNAs (vsiRNAs) to restrict virus infection by RNA interference (RNAi) is a recently identified mammalian immune response to several RNA viruses, which cause important human diseases such as influenza and Zika virus. However, little is known about Dicer processing of viral double-stranded RNA replicative intermediates (dsRNA-vRIs) in mammalian somatic cells. Here we show that infected somatic cells produced more influenza vsiRNAs than cellular microRNAs when both were produced by human Dicer expressed de novo, indicating that dsRNA-vRIs are not poor Dicer substrates as previously proposed according to in vitro Dicer processing of synthetic long dsRNA. We report the first evidence both for canonical vsiRNA production during wild-type Nodamura virus infection and direct vsiRNA sequestration by its RNAi suppressor protein B2 in two strains of suckling mice. Moreover, Sindbis virus (SINV) accumulation in vivo was decreased by prior production of SINV-targeting vsiRNAs triggered by infection and increased by heterologous expression of B2 in cis from SINV genome, indicating an antiviral function for the induced RNAi response. These findings reveal that unlike artificial long dsRNA, dsRNA-vRIs made during authentic infection of mature somatic cells are efficiently processed by Dicer into vsiRNAs to direct antiviral RNAi. Interestingly, Dicer processing of dsRNA-vRIs into vsiRNAs was inhibited by LGP2 (laboratory of genetics and physiology 2), which was encoded by an interferon-stimulated gene (ISG) shown recently to inhibit Dicer processing of artificial long dsRNA in cell culture. Our work thus further suggests negative modulation of antiviral RNAi by a known ISG from the interferon response.