Degradation of WTAP blocks antiviral responses by reducing the m6A levels of IRF3 and IFNAR1 mRNA

N ⁶‐methyladenosine (m⁶A) is a chemical modification present in multiple RNA species and is most abundant in mRNAs. Studies on m⁶A reveal its comprehensive roles in almost every aspect of mRNA metabolism, as well as in a variety of physiological processes. Although some recent discoveries indicate that m⁶A can affect the life cycles of numerous viruses as well as the cellular antiviral immune response, the roles of m⁶A modification in type I interferon (IFN‐I) signaling are still largely unknown. Here, we reveal that WT1‐associated protein (WTAP), one of the m⁶A “writers”, is degraded via the ubiquitination‐proteasome pathway upon activation of IFN‐I signaling. With the degradation of WTAP, the m⁶A levels of IFN‐regulatory factor 3 (IRF3) and interferon alpha/beta receptor subunit 1 (IFNAR1) mRNAs are reduced, leading to translational suppression of IRF3 and instability of IFNAR1 mRNA. Thus, the WTAP‐IRF3/IFNAR1 axis may serve as negative feedback pathway to fine‐tune the activation of IFN‐I signaling, which highlights the roles of m⁶A in the antiviral response by dictating the fate of mRNAs associated with IFN‐I signaling.     

Intercellular protein-protein interactions at synapses

Chemical synapses are asymmetric intercellular junctions through which neurons send nerve impulses to communicate with other neurons or excitable cells. The appropriate formation of synapses, both spatially and temporally, is essential for brain function and depends on the intercellular protein-protein interactions of cell adhesion molecules (CAMs) at synaptic clefts. The CAM proteins link pre- and post-synaptic sites, and play essential roles in promoting synapse formation and maturation, maintaining synapse number and type, accumulating neurotransmitter receptors and ion channels, controlling neuronal differentiation, and even regulating synaptic plasticity directly. Alteration of the interactions of CAMs leads to structural and functional impairments, which results in many neurological disorders, such as autism, Alzheimer’s disease and schizophrenia. Therefore, it is crucial to understand the functions of CAMs during development and in the mature neural system, as well as in the pathogenesis of some neurological disorders. Here, we review the function of the major classes of CAMs, and how dysfunction of CAMs relates to several neurological disorders.     

四个鲫鱼品系线粒体DNA的限制性酶切分析

用差速离心和核酸酶消化法从红鲫 (C auratusredvar .)、湘鲫 [F1hybridsofredcruciancarp (♀ )×commoncarp (♂ ) ]、野鲫 (C auratusauratus)和白鲫 (C auratuscuvieri)的肝组织及白鲫的卵巢中提取和纯化线粒体DNA。用 9种内切酶 (EcoRⅠ、HindⅢ、PstⅠ、BglⅡ、BamHⅠ、XhoⅠ、XbaⅠ、SalⅠ和KpnⅠ )进行单酶酶解 ,经琼脂糖凝胶电泳分析 ,检测出PstⅠ、KpnⅠ和BglⅡ 3种酶在品系间存在限制性片段长度多态性 ,但并未检测出品系内的限制性片段长度多态性。计算出红鲫、湘鲫、白鲫和野鲫的mtDNA大小分别约为 16 19、 16 0 2、 16 6 0和 16 0 6kb。根据限制性酶切片段共享度 ,计算出 4个品系间的遗传距离 ,结果表明存在直接亲缘关系的红鲫与湘鲫之间的遗传差异最小 ,证实了红鲫与子代湘鲫之间mtDNA遵循母系遗传的特性。     

Effect of dexamethasone on the expression of p34(cdc2) and cyclin B1 in pig oocytes in vitro

The meiotic division in oocytes is arrested in the G2 phase of the cell cycle. Resumption of meiosis, also known as oocyte maturation, entails a G2 to M transition. At the G2-M boundary, maturation promoting factor (MPF) activation is usually induced via several ways, including tyrosine dephosphorylation of p34(cdc2) and synthesis of cyclin B according to cell type and species. Previous studies in our laboratory demonstrated that glucocorticoids directly inhibit the meiotic maturation of pig oocytes in vitro. The aim of this study was therefore to investigate the influence of glucocorticoids on the expression of p34(cdc2) and cyclin B1 in resumption of meiosis of pig oocytes. We detected the relative levels and association of p34(cdc2) and cyclin B1. Isolated cumulus-enclosed oocytes were cultured in Waymouth MB752/1 medium supplemented with sodium pyruvate (50 microgram/ml), LH (0.5 microgram/ml), FSH (0.5 microgram/ml), and estradiol-17beta (1 microgram/ml) in the presence or absence of dexamethasone (DEX) for 24 hr; they then were cultured without hormonal supplements in the presence or absence of DEX for an additional 24 hr. We found that cyclin B1, as well as p34(cdc2), was already present in fully grown G2-arrested pig oocytes when removed from the follicle. In these oocytes, cyclin B1 and p34(cdc2) were already associated in complex. Treatment with DEX at concentrations of 1 microgram/ml or above decreased the level of cyclin B1, but had no effect on the level of p34(cdc2). The exposure of oocytes to DEX also decreased the amount of complexed p34(cdc2)-cyclin B1. These findings suggest that the inhibitory action of DEX on meiotic maturation could be due, at least in part, to the reduced amount of p34(cdc2)-cyclin B1 complex.     

Altered G protein-coupling functions of RNA editing isoform and splicing variant serotonin2C receptors

Different isoforms of serotonin subtype 2C receptor (5-HT(2C)R) with altered G protein-coupling efficacy are generated by RNA editing, which converts genomically encoded adenosine residues into inosines. In combination, editing of five sites all located within the second intracellular loop region of 5-HT(2C)R mRNA changes the gene-encoded Ile, Asn, and Ile at positions 156, 158, and 160, respectively. We analyzed the G protein-coupling functions of previously unreported editing isoform receptors. An approximately 13-fold reduction in the agonist potency for G protein-coupling stimulation as well as a significantly reduced basal level activity was observed with the thalamus-specific isoform carrying Ile156, Gly158, and Val160 (5-HT(2C)R-IGV). In contrast, the agonist was four- to five-fold less potent with 5-HT(2C)R-MSV and -IDV, detected in the amygdala and choroid plexus, respectively, indicating a dominant role for the amino acid residue at position 158 in receptor functions. We also identified a splicing variant receptor with a truncated C terminus that displayed no ligand binding capacity or G protein-coupling activity. Examination of the alternatively spliced RNA encoding this truncated receptor suggests that editing of this variant RNA occurs after completion of splicing, resulting in complete editing at all five sites.     

The RING finger ATPase Rad5p of Saccharomyces cerevisiae contributes to DNA double-strand break repair in a ubiquitin-independent manner

Tolerance to replication-blocking DNA lesions is achieved by means of ubiquitylation of PCNA, the processivity clamp for replicative DNA polymerases, by components of the RAD6 pathway. In the yeast Saccharomyces cerevisiae the ubiquitin ligase (E3) responsible for polyubiquitylation of the clamp is the RING finger protein Rad5p. Interestingly, the RING finger, responsible for the protein's E3 activity, is embedded in a conserved DNA-dependent ATPase domain common to helicases and chromatin remodeling factors of the SWI/SNF family. Here, we demonstrate that the Rad5p ATPase domain provides the basis for a function of the protein in DNA double-strand break repair via a RAD52- and Ku-independent pathway mediated by the Mre11/Rad50/Xrs2 protein complex. This activity is distinct and separable from the contribution of the RING domain to ubiquitin conjugation to PCNA. Moreover, we show that the Rad5 protein physically associates with the single-stranded DNA regions at a processed double-strand break in vivo. Our observations suggest that Rad5p is a multifunctional protein that—by means of independent enzymatic activities inherent in its RING and ATPase domains—plays a modulating role in the coordination of repair events and replication fork progression in response to various different types of DNA lesions.     

Multiple TLRs are expressed in human cholangiocytes and mediate host epithelial defense responses to Cryptosporidium parvum via activation of NF-kappaB

Infection of epithelial cells by Cryptosporidium parvum triggers a variety of host-cell innate and adaptive immune responses including release of cytokines/chemokines and up-regulation of antimicrobial peptides. The mechanisms that trigger these host-cell responses are unclear. Thus, we evaluated the role of TLRs in host-cell responses during C. parvum infection of cultured human biliary epithelia (i.e., cholangiocytes). We found that normal human cholangiocytes express all known TLRs. C. parvum infection of cultured cholangiocytes induces the selective recruitment of TLR2 and TLR4 to the infection sites. Activation of several downstream effectors of TLRs including IL-1R-associated kinase, p-38, and NF-kappaB was detected in infected cells. Transfection of cholangiocytes with dominant-negative mutants of TLR2 and TLR4, as well as the adaptor molecule myeloid differentiation protein 88 (MyD88), inhibited C. parvum-induced activation of IL-1R-associated kinase, p-38, and NF-kappaB. Short-interfering RNA to TLR2, TLR4, and MyD88 also blocked C. parvum-induced NF-kappaB activation. Moreover, C. parvum selectively up-regulated human beta-defensin-2 in directly infected cells, and inhibition of TLR2 and TLR4 signals or NF-kappaB activation were each associated with a reduction of C. parvum-induced human beta-defensin-2 expression. A significantly higher number of parasites were detected in cells transfected with a MyD88 dominant-negative mutant than in the control cells at 48-96 h after initial exposure to parasites, suggesting MyD88-deficient cells were more susceptible to infection. These findings demonstrate that cholangiocytes express a variety of TLRs, and suggest that TLR2 and TLR4 mediate cholangiocyte defense responses to C. parvum via activation of NF-kappaB.     

The SNPlex genotyping system: a flexible and scalable platform for SNP genotyping.

We developed the SNPlex Genotyping System to address the need for accurate genotyping data, high sample throughput, study design flexibility, and cost efficiency. The system uses oligonucleotide ligation/polymerase chain reaction and capillary electrophoresis to analyze bi-allelic single nucleotide polymorphism genotypes. It is well suited for single nucleotide polymorphism genotyping efforts in which throughput and cost efficiency are essential. The SNPlex Genotyping System offers a high degree of flexibility and scalability, allowing the selection of custom-defined sets of SNPs for medium- to high-throughput genotyping projects. It is therefore suitable for a broad range of study designs. In this article we describe the principle and applications of the SNPlex Genotyping System, as well as a set of single nucleotide polymorphism selection tools and validated assay resources that accelerate the assay design process. We developed the control pool, an oligonucleotide ligation probe set for training and quality-control purposes, which interrogates 48 SNPs simultaneously. We present performance data from this control pool obtained by testing genomic DNA samples from 44 individuals. in addition, we present data from a study that analyzed 521 SNPs in 92 individuals. Combined, both studies show the SNPlex Genotyping system to have a 99.32% overall call rate, 99.95% precision, and 99.84% concordance with genotypes analyzed by TaqMan probe-based assays. The SNPlex Genotyping System is an efficient and reliable tool for a broad range of genotyping applications, supported by applications for study design, data analysis, and data management.     

Identification and characterization of an NPV infection-related gene Bmsop2 in Bombyx mori L.

Abstract:  Using the fluorescent differential display technique, we analysed the differential expression of genes related to Bombyx mori nuclear polyhedrosis virus (BmNPV) resistance. Silkworm strains studied included the highly resistant strain NB, highly susceptible strain 306 and near-isogenic line 306NNZZ. One novel gene was identified and named Bmsop2 for its high similarity with the Sop2 protein of other species. It was identified to be linked to BmNPV susceptibility by Northern blotting and real-time polymerase chain reaction. The results indicated that it was actively expressed in midguts of strains 306, NB and the eighth generation of backcross (BC₈) of strain 306NNZZ which had been treated with BmNPV. But the expression level was low in the midguts of the control. In the mean time, the expression of Bmsop2 was the highest in strain 306 treated with BmNPV while it was the lowest in strain 306 not treated with BmNPV. Our study showed that Bmsop2 is a differentially expressed gene in strains NB, 306 and 306NNZZ which have different levels of resistance to BmNPV.     

Cholera toxin B protein in transgenic tomato fruit induces systemic immune response in mice

Cholera toxin B (CTB) subunit is a well-characterized antigen against cholera. Transgenic plants can offer an inexpensive and safe source of edible CTB vaccine and may be one of the best candidates for the production of plant vaccines. The present study aimed to develop transgenic tomato expressing CTB protein, especially in the ripening tomato fruit under the control of the tomato fruit-specific E8 promoter by using Agrobacterium-mediated transformation. Transgenic plants were selected using PCR and Southern blot analysis. Exogenous protein extracted from leaf, stem, and fruit tissues of transgenic plants was detected by ELISA and Western blot analysis, showing specific expression in the ripening fruit, with the highest amount of CTB protein being 0.081% of total soluble protein. Gavage of mice with ripe transgenic tomato fruits induced both serum and mucosal CTB specific antibodies. These results demonstrate the immunogenicity of the CTB protein in transgenic tomato and provide a considerable basis for exploring the utilization of CTB in the development of tomato-based edible vaccine against cholera. The rCTB antigen resulted in much lower antibody titers than an equal amount of exgenous CTB in trangenic fruits, suggesting the protective effect of the fibrous tissue of the fruit to the exogenous CTB protein against the degradation of protease in the digestive tracts of mice. Xiao-Ling Jiang and Zhu-Mei He contributed equally to this work.