Harnessing ZIKV NS2A RNA for alleviating acute hepatitis and cytokine release storm by targeting translation machinery.

BACKGROUND AND AIMS: Hyperactivated inflammatory responses induced by cytokine release syndrome (CRS) are the primary causes of tissue damage and even death. The translation process is precisely regulated to control the production of proinflammatory cytokines. However, it is largely unknown whether targeting translation can effectively limit the hyperactivated inflammatory responses during acute hepatitis and graft-versus-host disease (GVHD). APPROACH AND RESULTS: By using in vitro translation and cellular overexpression systems, we have found that the non-structural protein gene NS2A of Zika virus (ZIKV) functions as RNA molecules to suppress the translation of both ectopic genes and endogenous proinflammatory cytokines. Mechanistically, results from RNA pulldown and co-immunoprecipitation (Co-IP) assays have demonstrated that NS2A RNA interacts with the translation initiation factor eIF2α to disrupt the dynamic balance of the eIF2/eIF2B complex and translation initiation, which is the rate-limiting step during the translation process. In the acetaminophen (APAP)-, LPS/D-galactosamine (D-GalN)-, viral infection-induced acute hepatitis, and GVHD mouse models, mice with myeloid cell-specific knock-in of NS2A show decreased levels of serum proinflammatory cytokines and reduced tissue damage. CONCLUSIONS: ZIKV NS2A dampens the production of proinflammatory cytokines and alleviates inflammatory injuries by interfering translation process as RNA molecules, which suggests that NS2A RNA is potentially used to treat numerous acute inflammatory diseases characterized by CRS.

Molecular consequences of acute versus chronic CDK12 loss in prostate carcinoma nominates distinct therapeutic strategies.

Genomic loss of the transcriptional kinase CDK12 occurs in ~6% of metastatic castration-resistant prostate cancers (mCRPC) and correlates with poor patient outcomes. Prior studies demonstrate that acute CDK12 loss confers a homologous recombination (HR) deficiency (HRd) phenotype via premature intronic polyadenylation (IPA) of key HR pathway genes, including ATM. However, mCRPC patients have not demonstrated benefit from therapies that exploit HRd such as inhibitors of polyADP ribose polymerase (PARP). Based on this discordance, we sought to test the hypothesis that an HRd phenotype is primarily a consequence of acute CDK12 loss and the effect is greatly diminished in prostate cancers adapted to CDK12 loss. Analyses of whole genome sequences (WGS) and RNA sequences (RNAseq) of human mCRPCs determined that tumors with biallelic CDK12 alterations (CDK12 BAL ) lack genomic scar signatures indicative of HRd, despite carrying bi-allelic loss and the appearance of the hallmark tandem-duplicator phenotype (TDP). Experiments confirmed that acute CDK12 inhibition resulted in aberrant polyadenylation and downregulation of long genes (including BRCA1 and BRCA2) but such effects were modest or absent in tumors adapted to chronic CDK12 BAL . One key exception was ATM, which did retain transcript shortening and reduced protein expression in the adapted CDK12 BAL models. However, CDK12 BAL cells demonstrated intact HR as measured by RAD51 foci formation following irradiation. CDK12 BAL cells showed a vulnerability to targeting of CDK13 by sgRNA or CDK12/13 inhibitors and in vivo treatment of prostate cancer xenograft lines showed that tumors with CDK12 BAL responded to the CDK12/13 inhibitor SR4835, while CDK12-intact lines did not. Collectively, these studies show that aberrant polyadenylation and long HR gene downregulation is primarily a consequence of acute CDK12 deficiency, which is largely compensated for in cells that have adapted to CDK12 loss. These results provide an explanation for why PARPi monotherapy has thus far failed to consistently benefit patients with CDK12 alterations, though alternate therapies that target CDK13 or transcription are candidates for future research and testing.

Characterization of metallothionein genes from Broussonetia papyrifera: metal binding and heavy metal tolerance mechanisms.

BACKGROUND: Broussonetia papyrifera is an economically significant tree with high utilization value, yet its cultivation is often constrained by soil contamination with heavy metals (HMs). Effective scientific cultivation management, which enhances the yield and quality of B. papyrifera, necessitates an understanding of its regulatory mechanisms in response to HM stress. RESULTS: Twelve Metallothionein (MT) genes were identified in B. papyrifera. Their open reading frames ranged from 186 to 372 bp, encoding proteins of 61 to 123 amino acids with molecular weights between 15,473.77 and 29,546.96 Da, and theoretical isoelectric points from 5.24 to 5.32. Phylogenetic analysis classified these BpMTs into three subclasses: MT1, MT2, and MT3, with MT2 containing seven members and MT3 only one. The expression of most BpMT genes was inducible by Cd, Mn, Cu, Zn, and abscisic acid (ABA) treatments, particularly BpMT2e, BpMT2d, BpMT2c, and BpMT1c, which showed significant responses and warrant further study. Yeast cells expressing these BpMT genes exhibited enhanced tolerance to Cd, Mn, Cu, and Zn stresses compared to control cells. Yeasts harboring BpMT1c, BpMT2e, and BpMT2d demonstrated higher accumulation of Cd, Cu, Mn, and Zn, suggesting a chelation and binding capacity of BpMTs towards HMs. Site-directed mutagenesis of cysteine (Cys) residues indicated that mutations in the C domain of type 1 BpMT led to increased sensitivity to HMs and reduced HM accumulation in yeast cells; While in type 2 BpMTs, the contribution of N and C domain to HMs' chelation possibly corelated to the quantity of Cys residues. CONCLUSION: The BpMT genes are crucial in responding to diverse HM stresses and are involved in ABA signaling. The Cys-rich domains of BpMTs are pivotal for HM tolerance and chelation. This study offers new insights into the structure-function relationships and metal-binding capabilities of type-1 and - 2 plant MTs, enhancing our understanding of their roles in plant adaptation to HM stresses.

diABZI and poly(I:C) inhibit osteoclastic bone resorption by inducing IRF7 and IFIT3.

Type I interferons (IFN-I) are pleiotropic factors endowed with multiple activities that play important roles in innate and adaptive immunity. Although many studies indicate that IFN-I inducers exert favorable effects on broad-spectrum antivirus, immunomodulation, and anti-tumor activities by inducing endogenous IFN-I and IFN-stimulated genes, their function in bone homeostasis still needs further exploration. Here, our study demonstrates 2 distinct IFN-I inducers, diABZI and poly(I:C), as potential therapeutics to alleviate osteolysis and osteoporosis. First, IFN-I inducers suppress the genes that control osteoclast (OC) differentiation and activity in vitro. Moreover, diABZI alleviates bone loss in Ti particle-induced osteolysis and ovariectomized -induced osteoporosis in vivo by inhibiting OC differentiation and function. In addition, the inhibitory effects of IFN-I inducers on OC differentiation are not observed in macrophages derived from Ifnar1-/-mice, which indicate that the suppressive effect of IFN-I inducers on OC is IFNAR-dependent. Mechanistically, RNAi-mediated silencing of IRF7 and IFIT3 in OC precursors impairs the suppressive effect of the IFN-I inducers on OC differentiation. Taken together, these results demonstrate that IFN-I inducers play a protective role in bone turnover by limiting osteoclastogenesis and bone resorption through the induction of OC-specific mediators via the IFN-I signaling pathway.

OCs are responsible for bone resorption, and their excessive differentiation and enhanced activity will lead to bone resorption diseases such as osteoporosis and osteolysis. Here, our study demonstrates 2 distinct IFN-I inducers, diABZI and poly(I:C), as potential therapeutics to alleviate osteolysis and osteoporosis. IFN-I inducers suppress OC differentiation, and particularly diABZI alleviates bone loss in osteolysis and osteoporosis mouse models. Taken together, IFN-I inducers play a protective role in bone turnover by limiting osteoclastogenesis and bone resorption through the induction of OC-specific mediators via the IFN-I signaling pathway. Our in-depth and comprehensive discovery of the IFN-I inducer would provide new insight into OC biology and therapeutic targets for osteoclastic bone resorption diseases.

Housekeeping U1 snRNA facilitates antiviral innate immunity by promoting TRIM25-mediated RIG-I activation.

U1 small nuclear RNA (snRNA) is an abundant and evolutionarily conserved 164-nucleotide RNA species that functions in pre-mRNA splicing, and it is considered to be a housekeeping non-coding RNA. However, the role of U1 snRNA in regulating host antiviral immunity remains largely unexplored. Here, we find that RNVU1-18, a U1 pseudogene, is significantly upregulated in the host infected with RNA viruses, including influenza and respiratory syncytial virus. Overexpression of U1 snRNA protects cells against RNA viruses, while knockdown of U1 snRNA leads to more viral burden in vitro and in vivo. Knockout of RNVU1-18 is sufficient to impair the type I interferon-dependent antiviral innate immunity. U1 snRNA is required to fully activate the retinoic acid-inducible gene I (RIG-I)-dependent antiviral signaling, since it interacts with tripartite motif 25 (TRIM25) and enhances the RIG-I-TRIM25 interaction to trigger K63-linked ubiquitination of RIG-I. Our study reveals the important role of housekeeping U1 snRNA in regulating host antiviral innate immunity and restricting RNA virus infection.

Targeting the fibroblast growth factor pathway in molecular subtypes of castration-resistant prostate cancer.

BACKGROUND: Androgen receptor (AR) pathway inhibition remains the cornerstone for prostate cancer therapies. However, castration-resistant prostate cancer (CRPC) tumors can resist AR signaling inhibitors through AR amplification and AR splice variants in AR-positive CRPC (ARPC), and conversion to AR-null phenotypes, such as double-negative prostate cancer (DNPC) and small cell or neuroendocrine prostate cancer (SCNPC). We have shown previously that DNPC can bypass AR-dependence through fibroblast growth factor receptor (FGFR) signaling. However, the role of the FGFR pathway in other CRPC phenotypes has not been elucidated. METHODS: RNA-Seq analysis was conducted on patient metastases, LuCaP patient-derived xenograft (PDX) models, and CRPC cell lines. Cell lines (C4-2B, VCaP, and 22Rv1) and ex vivo LuCaP PDX tumor cells were treated with enzalutamide (ENZA) and FGFR inhibitors (FGFRi) alone or in combination and sensitivity was determined using cell viability assays. In vivo efficacy of FGFRi in ARPC, DNPC, and SCNPC were evaluated using PDX models. RESULTS: RNA-Seq analysis of FGFR signaling in metastatic specimens, LuCaP PDX models, and CRPC cell lines revealed significant FGF pathway activation in AR-low PC (ARLPC), DNPC, and SCNPC tumors. In vitro/ex vivo analysis of erdafitinib and CH5183284 demonstrated robust and moderate growth suppression of ARPC, respectively. In vivo studies using four ARPC PDX models showed that combination ENZA and CH5183284 significantly suppressed tumor growth. Additional in vivo studies using four ARPC PDX models revealed that erdafitinib monotherapy was as effective as ENZA in suppressing tumor growth, and there was limited combination benefit. Furthermore, two of three DNPC models and two of four SCNPC models responded to CH5183284 monotherapy, suggesting FGFRi responses were model dependent. RNA-Seq and gene set enrichment analysis of end-of-study ARPC tumors treated with FGFRi displayed decreased expression of E2F and MYC target genes and suppressed G2M checkpoint genes, whereas end-of-study SCNPC tumors had heterogeneous transcriptional responses. CONCLUSIONS: Although FGFRi treatments suppressed tumor growth across CRPC phenotypes, our analyses did not identify a single pathway or biomarker that would identify tumor response to FGFRi. This is very likely due to the array of FGFR1-4 expression and tumor phenotypes present in CRPC. Nevertheless, our data nominate the FGFR pathway as a clinically actionable target that promotes tumor growth in diverse phenotypes of treatment-refractory metastatic CRPC.

Prostate lineage-specific metabolism governs luminal differentiation and response to antiandrogen treatment.

Lineage transitions are a central feature of prostate development, tumourigenesis and treatment resistance. While epigenetic changes are well known to drive prostate lineage transitions, it remains unclear how upstream metabolic signalling contributes to the regulation of prostate epithelial identity. To fill this gap, we developed an approach to perform metabolomics on primary prostate epithelial cells. Using this approach, we discovered that the basal and luminal cells of the prostate exhibit distinct metabolomes and nutrient utilization patterns. Furthermore, basal-to-luminal differentiation is accompanied by increased pyruvate oxidation. We establish the mitochondrial pyruvate carrier and subsequent lactate accumulation as regulators of prostate luminal identity. Inhibition of the mitochondrial pyruvate carrier or supplementation with exogenous lactate results in large-scale chromatin remodelling, influencing both lineage-specific transcription factors and response to antiandrogen treatment. These results establish reciprocal regulation of metabolism and prostate epithelial lineage identity.

SND1 binds to ERG and promotes tumor growth in genetic mouse models of prostate cancer.

SND1 and MTDH are known to promote cancer and therapy resistance, but their mechanisms and interactions with other oncogenes remain unclear. Here, we show that oncoprotein ERG interacts with SND1/MTDH complex through SND1's Tudor domain. ERG, an ETS-domain transcription factor, is overexpressed in many prostate cancers. Knocking down SND1 in human prostate epithelial cells, especially those overexpressing ERG, negatively impacts cell proliferation. Transcriptional analysis shows substantial overlap in genes regulated by ERG and SND1. Mechanistically, we show that ERG promotes nuclear localization of SND1/MTDH. Forced nuclear localization of SND1 prominently increases its growth promoting function irrespective of ERG expression. In mice, prostate-specific Snd1 deletion reduces cancer growth and tumor burden in a prostate cancer model (PB-Cre/Ptenflox/flox/ERG mice), Moreover, we find a significant overlap between prostate transcriptional signatures of ERG and SND1. These findings highlight SND1's crucial role in prostate tumorigenesis, suggesting SND1 as a potential therapeutic target in prostate cancer.

Concurrent Targeting of HDAC and PI3K to Overcome Phenotypic Heterogeneity of Castration-resistant and Neuroendocrine Prostate Cancers.

Castration-resistant prostate cancer (CRPC) consists of multiple phenotypic subtypes including androgen receptor (AR)-active prostate cancer (ARPC) and neuroendocrine prostate cancer (NEPC). Tumor cells with these phenotypes can coexist between metastases within a patient and within an individual tumor. Treatments that are effective across CRPC subtypes are currently lacking. Histone deacetylation is crucial for the regulation of chromatin structure and maintenance of cancer cell state and activation of the PI3K/AKT/mTOR signaling cascade is a tumor growth-promoting pathway. We therefore investigated combined targeting of histone deacetylase (HDAC) and PI3K using a rationally designed dual inhibitor, fimepinostat, in CRPC subtypes in vitro and in vivo. Dual HDAC1/2 and PI3K/AKT pathway inhibition by fimepinostat led to robust tumor growth inhibition in both ARPC and NEPC models including cell line- and patient-derived xenografts. HDAC1/2 inhibition combined with PI3K/AKT inhibition was more effective than targeting each pathway alone, producing growth inhibitory effects through cell-cycle inhibition and apoptosis. Molecular profiling revealed on-target effects of combined HDAC1/2 and PI3K/AKT inhibition independent of tumor phenotype. Fimepinostat therapy was also associated with the suppression of lineage transcription factors including AR in ARPC and Achaete-scute homolog 1 (ASCL1) in NEPC. Together, these results indicate that fimepinostat represents a novel therapeutic that may be effective against both ARPC and NEPC through CRPC subtype-dependent and -independent mechanisms. SIGNIFICANCE: CRPC is a heterogeneous disease constituting multiple phenotypic subtypes that often co-occur within tumors or across metastases in patients. Existing targeted therapies for CRPC do not take this into account. Here we show that fimepinostat, a dual HDAC1/2 and PI3K/AKT inhibitor investigated clinically in other cancer types but not prostate cancer, may overcome this heterogeneity by effectively inhibiting both ARPC and NEPC subtypes of CRPC.

Supraphysiological Androgens Promote the Tumor Suppressive Activity of the Androgen Receptor through cMYC Repression and Recruitment of the DREAM Complex.

The androgen receptor (AR) pathway regulates key cell survival programs in prostate epithelium. The AR represents a near-universal driver and therapeutic vulnerability in metastatic prostate cancer, and targeting AR has a remarkable therapeutic index. Though most approaches directed toward AR focus on inhibiting AR signaling, laboratory and now clinical data have shown that high dose, supraphysiological androgen treatment (SPA) results in growth repression and improved outcomes in subsets of patients with prostate cancer. A better understanding of the mechanisms contributing to SPA response and resistance could help guide patient selection and combination therapies to improve efficacy. To characterize SPA signaling, we integrated metrics of gene expression changes induced by SPA together with cistrome data and protein-interactomes. These analyses indicated that the dimerization partner, RB-like, E2F, and multivulval class B (DREAM) complex mediates growth repression and downregulation of E2F targets in response to SPA. Notably, prostate cancers with complete genomic loss of RB1 responded to SPA treatment, whereas loss of DREAM complex components such as RBL1/2 promoted resistance. Overexpression of MYC resulted in complete resistance to SPA and attenuated the SPA/AR-mediated repression of E2F target genes. These findings support a model of SPA-mediated growth repression that relies on the negative regulation of MYC by AR leading to repression of E2F1 signaling via the DREAM complex. The integrity of MYC signaling and DREAM complex assembly may consequently serve as determinants of SPA responses and as pathways mediating SPA resistance. SIGNIFICANCE: Determining the molecular pathways by which supraphysiological androgens promote growth arrest and treatment responses in prostate cancer provides opportunities for biomarker-selected clinical trials and the development of strategies to augment responses.

A genome-wide CRISPR screen in human prostate cancer cells reveals drivers of macrophage-mediated cell killing and positions AR as a tumor-intrinsic immunomodulator.

The crosstalk between prostate cancer (PCa) cells and the tumor microenvironment plays a pivotal role in disease progression and metastasis and could provide novel opportunities for patient treatment. Macrophages are the most abundant immune cells in the prostate tumor microenvironment (TME) and are capable of killing tumor cells. To identify genes in the tumor cells that are critical for macrophage-mediated killing, we performed a genome-wide co-culture CRISPR screen and identified AR, PRKCD, and multiple components of the NF-κB pathway as hits, whose expression in the tumor cell are essential for being targeted and killed by macrophages. These data position AR signaling as an immunomodulator, and confirmed by androgen-deprivation experiments, that rendered hormone-deprived tumor cells resistant to macrophage-mediated killing. Proteomic analyses showed a downregulation of oxidative phosphorylation in the PRKCD- and IKBKG-KO cells compared to the control, suggesting impaired mitochondrial function, which was confirmed by electron microscopy analyses. Furthermore, phosphoproteomic analyses revealed that all hits impaired ferroptosis signaling, which was validated transcriptionally using samples from a neoadjuvant clinical trial with the AR-inhibitor enzalutamide. Collectively, our data demonstrate that AR functions together with the PRKCD and the NF-κB pathway to evade macrophage-mediated killing. As hormonal intervention represents the mainstay therapy for treatment of prostate cancer patients, our findings may have direct implications and provide a plausible explanation for the clinically observed persistence of tumor cells despite androgen deprivation therapy.

XAF1 Protects Host against Emerging RNA Viruses by Stabilizing IRF1-Dependent Antiviral Immunity.

XIAP-associated factor 1 (XAF1) is an interferon (IFN)-stimulated gene (ISG) that enhances IFN-induced apoptosis. However, it is unexplored whether XAF1 is essential for the host fighting against invaded viruses. Here, we find that XAF1 is significantly upregulated in the host cells infected with emerging RNA viruses, including influenza, Zika virus (ZIKV), and SARS-CoV-2. IFN regulatory factor 1 (IRF1), a key transcription factor in immune cells, determines the induction of XAF1 during antiviral immunity. Ectopic expression of XAF1 protects host cells against various RNA viruses independent of apoptosis. Knockout of XAF1 attenuates host antiviral innate immunity in vitro and in vivo, which leads to more severe lung injuries and higher mortality in the influenza infection mouse model. XAF1 stabilizes IRF1 protein by antagonizing the CHIP-mediated degradation of IRF1, thus inducing more antiviral IRF1 target genes, including DDX58, DDX60, MX1, and OAS2. Our study has described a protective role of XAF1 in the host antiviral innate immunity against RNA viruses. We have also elucidated the molecular mechanism that IRF1 and XAF1 form a positive feedback loop to induce rapid and robust antiviral immunity. IMPORTANCE Rapid and robust induction of antiviral genes is essential for the host to clear the invaded viruses. In addition to the IRF3/7-IFN-I-STAT1 signaling axis, the XAF1-IRF1 positive feedback loop synergistically or independently drives the transcription of antiviral genes. Moreover, XAF1 is a sensitive and reliable gene that positively correlates with the viral infection, suggesting that XAF1 is a potential diagnostic marker for viral infectious diseases. In addition to the antitumor role, our study has shown that XAF1 is essential for antiviral immunity. XAF1 is not only a proapoptotic ISG, but it also stabilizes the master transcription factor IRF1 to induce antiviral genes. IRF1 directly binds to the IRF-Es of its target gene promoters and drives their transcriptions, which suggests a unique role of the XAF1-IRF1 loop in antiviral innate immunity, particularly in the host defect of IFN-I signaling such as invertebrates.

Identifying and expression analysis of WD40 transcription factors in walnut.

Walnut (Juglans regia L.) is an important woody oil plant and will be affected by abiotic and biological stress during its growth and development. The WD-repeat (WD40) protein is widely involved in plant growth, development, metabolism, and abiotic stress response. To explore the stress response mechanism of walnut, based on the complete sequencing results of the walnut genome, this study identified and analyzed the physiological, biochemical, genetic structure, and conservative protein motifs of 42 JrWD40 genes, whose expression to abnormal temperature were tested to predict the potential biological function. The results showed that the open reading frame (ORF) of theseWD40 genes were 807-2,460 bp, encoding peptides were 29,610.55-90,387.98 Da covering 268-819 amino acids, as well as 12-112 phosphorylation sites. JrWD40 proteins were highly conserved with four to five WD40 domains and shared certain similarity to WD40 proteins from Arabidopsis thaliana (L.) Heynh. JrWD40 genes can be induced to varying degrees by low and high temperature treatments. JrWD40-32, JrWD40-27, JrWD40-35, and JrWD40-21 are affected by high temperature more seriously and their expression levels are higher; while JrWD40-37, JrWD40-26, JrWD40-20, JrWD40-24, and other genes are inhibited under low temperature stress. JrWD40-40, JrWD40-28, and JrWD40-18 were first suppressed with low expression, while as the treatment time prolonging, the expression level was increased under cold condition. JrWD40-14, JrWD40-18, JrWD40-34, and JrWD40-3 displayed strong transcriptions response to both heat and cold stress. These results indicated that JrWD40 genes can participate in walnut adaptation to adversity and can be used as important candidates for walnut resistance molecular breeding.

Therapeutic Implications for Intrinsic Phenotype Classification of Metastatic Castration-Resistant Prostate Cancer.

PURPOSE: To determine whether metastatic castration-resistant prostate cancers (mCRPC) partition into molecular phenotypes corresponding to intrinsic differentiation states and ascertain whether these subtypes exhibit specific druggable features and associate with treatment outcomes. EXPERIMENTAL DESIGN: We used RNAseq, digital spatial profiling, and histological assessments from metastatic biopsies and patient-derived xenografts to segregate mCRPCs into subtypes defined by the PAM50 breast cancer classification algorithm. Subtype associations with treatment responses in preclinical models and patients were determined. RESULTS: Using the PAM50 algorithm, we partitioned 270 mCRPC tumors into LumA (42%), LumB (24%), and Basal (34%) subtypes with classification largely driven by proliferation rates and androgen receptor (AR) activity. Most neuroendocrine tumors classified as Basal. Pathways enriched in the LumA subtype include TGFß and NOTCH signaling. LumB subtype tumors were notable for elevated MYC activity. Basal subtype tumors exhibited elevated IL6-STAT3 signaling and features of adult stem cell states. In patients where multiple tumors were evaluated, the majority had concordant PAM50 subtype determination, though a subset exhibited marked inter- and intratumor heterogeneity, including divergent classifications between primary and metastatic sites. In preclinical models, LumA subtype tumors were highly responsive to androgen deprivation and docetaxel chemotherapy whereas Basal tumors were largely resistant. In clinical cohorts patients with Basal subtype tumors demonstrated a shorter time on treatment with AR signaling inhibitors and docetaxel relative to patients with luminal subtypes. CONCLUSIONS: Subtyping of mCRPC based on cell differentiation states has potential clinical utility for identifying patients with divergent expression of treatment targets and responses to systemic therapy.

Anti-apoptotic HAX-1 suppresses cell apoptosis by promoting c-Abl kinase-involved ROS clearance.

The anti-apoptotic protein HAX-1 has been proposed to modulate mitochondrial membrane potential, calcium signaling and actin remodeling. HAX-1 mutation or deficiency results in severe congenital neutropenia (SCN), loss of lymphocytes and neurological impairments by largely unknown mechanisms. Here, we demonstrate that the activation of c-Abl kinase in response to oxidative or genotoxic stress is dependent on HAX-1 association. Cellular reactive oxygen species (ROS) accumulation is inhibited by HAX-1-dependent c-Abl activation, which greatly contributes to the antiapoptotic role of HAX-1 in stress. HAX-1 (Q190X), a loss-of-function mutant responsible for SCN, fails to bind with and activate c-Abl, leading to dysregulated cellular ROS levels, damaged mitochondrial membrane potential and eventually apoptosis. The extensive apoptosis of lymphocytes and neurons in Hax-1-deficient mice could also be remarkably suppressed by c-Abl activation. These findings underline the important roles of ROS clearance in HAX-1-mediated anti-apoptosis by c-Abl kinase activation, providing new insight into the pathology and treatment of HAX-1-related hereditary disease or tumorigenesis.

LincRNA-EPS impairs host antiviral immunity by antagonizing viral RNA-PKR interaction.

LincRNA-EPS is an important regulator in inflammation. However, the role of lincRNA-EPS in the host response against viral infection is unexplored. Here, we show that lincRNA-EPS is downregulated in macrophages infected with different viruses including VSV, SeV, and HSV-1. Overexpression of lincRNA-EPS facilitates viral infection, while deficiency of lincRNA-EPS protects the host against viral infection in vitro and in vivo. LincRNA-EPS-/- macrophages show elevated expression of antiviral interferon-stimulated genes (ISGs) such as Mx1, Oas2, and Ifit2 at both basal and inducible levels. However, IFN-ß, the key upstream inducer of these ISGs, is downregulated in lincRNA-EPS-/- macrophages compared with control cells. RNA pulldown and mass spectrometry results indicate that lincRNA-EPS binds to PKR and antagonizes the viral RNA-PKR interaction. PKR activates STAT1 and induces antiviral ISGs independent of IFN-I induction. LincRNA-EPS inhibits PKR-STAT1-ISGs signaling and thus facilitates viral infection. Our study outlines an alternative antiviral pathway, with downregulation of lincRNA-EPS promoting the induction of PKR-STAT1-dependent ISGs, and reveals a potential therapeutic target for viral infectious diseases.

Walnut ethylene response factor JrERF2-2 interact with JrWRKY7 to regulate the GSTs in plant drought tolerance.

Juglans regia is a world-famous woody oil plant, whose yield and quality are affected by drought stress. Ethylene-responsive factors (ERFs) play vital role in plant stress response. In current study, to comprehend the walnut molecular mechanism of drought stress response, an ERF transcription factor was clarified from J. regia (JrERF2-2) and its potential function mechanism to drought was clarified. The results showed that JrERF2-2 could be induced significantly by drought. The transgenic Arabidopsis over-expression of JrERF2-2 displayed enhanced growth, antioxidant enzyme vitalities, reactive oxygen species scavenging and proline produce under drought stress. Especial the glutathione-S-transferase (GST) activity and most GST genes' transcription were elevated obviously. Yeast one-hybrid (Y1H) and co-transient expression (CTE) methods revealed that JrERF2-2 could recognize JrGST4, JrGST6, JrGST7, JrGST8, and JrGSTF8 by binding to GCC-box, and recognize JrGST11, JrGST12, and JrGSTN2 by binding to DRE motif. Meanwhile, the binding activity was strengthened by drought stress. Moreover, JrERF2-2 could interact with JrWRKY7 to promote plant drought tolerance; JrWRKY7 could also distinguish JrGST4, JrGST7, JrGST8, JrGST11, JrGST12, and JrGSTF8 via binding to W-Box motif. These results suggested that JrERF2-2 could effectively improve plant drought tolerance through interacting with JrWRKY7 to control the expression of GSTs. JrERF2-2 is a useful plant representative gene for drought response in molecular breeding.

IFI16 Isoforms with Cytoplasmic and Nuclear Locations Play Differential Roles in Recognizing Invaded DNA Viruses.

IFN-γ-inducible protein 16 (IFI16) recognizes viral DNAs from both nucleus-replicating viruses and cytoplasm-replicating viruses. Isoform 2 of IFI16 (IFI16-iso2) with nuclear localization sequence (NLS) has been studied extensively as a well-known DNA sensor. However, the characteristics and functions of other IFI16 isoforms are almost unknown. Here, we find that IFI16-iso1, with exactly the same length as IFI16-iso2, lacks the NLS and locates in the cytoplasm. To distinguish the functions of IFI16-iso1 and IFI16-iso2, we have developed novel nuclear viral DNA mimics that can be recognized by the nuclear DNA sensors, including IFI16-iso2 and hnRNPA2B1. The hexanucleotide motif 5'-AGTGTT-3' DNA form of the nuclear localization sequence (DNLS) effectively drives cytoplasmic viral DNA nuclear translocation. These nuclear viral DNA mimics potently induce IFN-ß and antiviral IFN-stimulated genes in human A549 cells, HEK293T cells, and mouse macrophages. The subcellular location difference of IFI16 isoforms determines their differential functions in recognizing viral DNA and activating type I IFN-dependent antiviral immunity. IFI16-iso1 preferentially colocalizes with cytoplasmic HSV60mer and cytoplasm-replicating vaccinia virus (VACV), whereas IFI16-iso2 mainly colocalizes with nuclear HSV60-DNLS and nucleus-replicating HSV-1. Compared with IFI16-iso2, IFI16-iso1 induces more transcription of IFN-ß and IFN-stimulated genes, as well as stronger antiviral immunity upon HSV60mer transfection or VACV infection. IFI16-iso2, with the ability of nuclear-cytoplasmic shuttling, clears both invaded HSV type 1 and VACV significantly. However, IFI16-iso2 induces more type I IFN-dependent antiviral immunity than IFI16-iso1 upon HSV60-DNLS transfection or HSV type 1 infection. Our study has developed potent agonists for nuclear DNA sensors and also has demonstrated that IFI16 isoforms with cytoplasmic and nuclear locations play differential roles in innate immunity against DNA viruses.

Genome-wide identification and expression analysis of ethylene responsive factor family transcription factors in Juglans regia.

BACKGROUND: Walnut is an important economic tree species with prominent economic value and ecological functions. However, in recent years, walnuts have become susceptible to drought stress, resulting in a decline in comprehensive benefits. Therefore, it is necessary to identify the regulatory molecular mechanism associated with walnut response to drought. In many plants, ethylene responsive factor (ERF) gene family plays important roles in response to biotic and abiotic stress, especial drought. Therefore, the identification and characterisation of walnut ERF genes will benefit walnut with regard to the clarification of drought response mechanism as well as the management, production, and quality of plantations. METHODS: 'ERF' was compared against the walnut transcriptome, and the JrERFs with a complete open reading frame (ORF) were identified by ORF Finder. The molecular weights, amino acid residues, and theoretical isoelectric point (pI) were predicted by ExPASy. The distribution of JrERFs in chromosome locations was determined based on walnut genome data from NCBI. The intron-exon structures and conserved domains were analysed using Gene Structure Display Server 2.0 and CD-Search, accordingly. Multi-sequence alignment and a phylogenetic tree were constructed by ClustalX2.1 and MEGA7, respectively. The conserved motifs were acquired using MEME. Total RNA was isolated using the cetyltrimethylammonium ammonium bromide (CTAB) method (Yang et al., 2018). Gene expression was determined by using real-time quantitative polymerase chain reaction (qRT-PCR) analysis and calculated according to the 2-ΔΔCT method (Livak & Schmittgen, 2001). RESULTS: A total of 44 JrERFs were identified from the walnut transcriptome, whose ORFs were 450-1,239 bp in length. The molecular weights of the JrERF proteins (consisting 149-412 amino acids) were 16.81-43.71 kDa, with pI ranging from 4.8 (JrERF11) to 9.89 (JrERF03). The JrERFs can be divided into six groups (B1-B6), and among the groups, B6 contained the most number of members. Each JrERF contained 1-6 motifs and each motif comprised 9-50 amino acids. Among the motifs, motif1, motif2, and motif3 were the most abundant. More than 40% of JrERFs were up-regulated continuously when subjected to ethephon (ETH), PEG6000, and PEG6000+ETH treatments. Of all the JrERFs, JrERF11 showed the highest expression. Therefore, we conclude that walnut ERF genes are highly conserved and involved in the regulation of drought response in the presence of ETH. JrERFs are possibly important candidate genes for molecular breeding; hence, the findings of this study provides the theoretical basis for further investigation of ERF genes in walnut and other species.

RNA Splicing Factors SRRM3 and SRRM4 Distinguish Molecular Phenotypes of Castration-Resistant Neuroendocrine Prostate Cancer.

Neuroendocrine (NE) differentiation in metastatic castration-resistant prostate cancer (mCRPC) is an increasingly common clinical feature arising from cellular plasticity. We recently characterized two mCRPC phenotypes with NE features: androgen receptor (AR)-positive NE-positive amphicrine prostate cancer (AMPC) and AR-negative small cell or neuroendocrine prostate cancer (SCNPC). Here, we interrogated the regulation of RE1-silencing transcription factor (REST), a transcriptional repressor of neuronal genes, and elucidated molecular programs driving AMPC and SCNPC biology. Analysis of prostate cancer cell lines, mCRPC specimens, and LuCaP patient-derived xenograft models detected alternative splicing of REST to REST4 and attenuated REST repressor activity in AMPC and SCNPC. The REST locus was also hypermethylated and REST expression was reduced in SCNPC. While serine/arginine repetitive matrix protein 4 (SRRM4) was previously implicated in alternative splicing of REST in mCRPC, we detected SRRM3 expression in REST4-positive, SRRM4-negative AMPC, and SCNPC. In CRPC cell lines, SRRM3 induced alternative splicing of REST to REST4 and exacerbated the expression of REST-repressed genes. Furthermore, SRRM3 and SRRM4 expression defined molecular subsets of AMPC and SCNPC across species and tumor types. Two AMPC phenotypes and three SCNPC phenotypes were characterized, denoted either by REST attenuation and ASCL1 activity or by progressive activation of neuronal transcription factor programs, respectively. These results nominate SRRM3 as the principal REST splicing factor expressed in early NE differentiation and provide a framework to molecularly classify diverse NE phenotypes in mCRPC. SIGNIFICANCE: This study identifies SRRM3 as a key inducer of cellular plasticity in prostate cancer with neuroendocrine features and delineates distinct neuroendocrine phenotypes to inform therapeutic development and precision medicine applications.