CCCH-type Zinc Finger Research Articles

A loss-of-function mutation in OsTZF5 confers sensitivity to low temperature and effects the growth and development in rice.

Rice (Oryza sativa L.) is highly sensitive to low temperatures, which can significantly reduce its production. Cold tolerance in rice is a complex trait regulated by multiple mechanisms. OsTZF5, a member of the CCCH-type zinc finger gene family in rice, has been previously reported that overexpressing OsTZF5 under the stress-responsive promoter can confer drought resistance. In this study, we showed that the loss of function mutants of OsTZF5 decreased seed germination rate and chilling tolerance in rice, and influencing normal growth and development. OsTZF5 is expressed in various parts of the rice plant, including roots, stems, leaves and inflorescences, with the highest expression levels observed in leaves. Additionally, the expression of OsTZF5 gene was influenced by various stress conditions and hormone treatments. OsTZF5 knock-out mutants exhibited significantly lower survival rates compared to the wild type (Zhonghua11, ZH11) after cold stress, as well as fewer tillers, lower thousand-grain weight, and reduced grain yield under normal conditions. Transcriptomic analyses revealed that the expression of cold stress-related genes was significantly down-regulated in OsTZF5 knock-out mutants compared to ZH11 after cold stress. This down-regulation likely contributes to the reduced cold stress tolerance observed in OsTZF5 knock-out mutants. Our findings suggest that OsTZF5 is a multifunctional gene that plays a crucial role in regulating cold stress in rice.

M6A methyltransferase ZC3H13 improves pulmonary fibrosis in mice through regulating Bax expression

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease. N6-methyladenosine (m6A) is a reversible RNA modification that was shown to be associated with IPF development. The present study aimed to explore the function and potential mechanism of the m6A methylation enzyme zinc finger CCCH-type containing 13 (ZC3H13) in IPF. In the study, bioinformatic screening yielded a differentially expressed m6A gene, ZC3H13, which was down-regulated in GEO microarrays, BLM-induced mouse models, and cellular models. Overexpression of ZC3H13 reduced histopathological damage of lung tissues in mice, mitigated fibrosis (including reduced α-SMA, collagen Ⅰ, and Vimentin levels, and elevated E-cadherin levels), decreased lung/body weight ratio and lung hydroxyproline levels, reduced oxidative stress (increased SOD activity and GSH-Px activity and decreased MDA levels), suppressed apoptosis within lung tissues and MLE-12 cells, promoted Bcl-2 expression, and inhibited Bax expression. Bax expression was found to be negatively correlated with ZC3H13 expression by correlation analysis. ZC3H13 could bind Bax mRNA and promote its m6A methylation through reading protein YTHDC1, thereby inhibiting its stability. Bax inhibition ameliorated BLM-induced MLE-12 cell dysfunction and partially abrogated the inhibition of MLE-12 cell function by ZC3H13 downregulation. In conclusion, m6A methyltransferase ZC3H13 impedes lung epithelial cell apoptosis and thus improves pulmonary fibrosis by promoting Bax mRNA m6A methylation and down-regulating Bax expression through reading protein YTHDC1.

ZC3H13 knockdown enhances the inhibitory effect of sevoflurane on gastric cancer cell malignancy by regulating the N6-methyladenosine modification of the lncRNA DLX6-AS1

Sevoflurane, an inhalation anesthetic, has been shown to suppress cancer development. In this study, we investigated the specific mechanisms involving sevoflurane, zinc-finger CCCH-type containing 13 (ZC3H13), and lncRNA DLX6-AS1 in gastric cancer (GC) progression, focusing on the N6-methyladenosine (m6A) modification of long non-coding RNAs (lncRNAs). We used quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analyses to measure the levels of ZC3H13 and lncRNA DLX6-AS1 in GC tissues and cells. Furthermore, we conducted Cell Counting Kit-8, colony formation, Transwell, and tumor xenograft assays to evaluate changes in GC cell malignancy following cell transfection and sevoflurane treatment. Additionally, actinomycin D, methylated RNA immunoprecipitation, and qRT-PCR assays were performed to examine the regulatory effects of ZC3H13 on the DLX6-AS1 m6A modification. We detected elevated levels of ZC3H13 in GC samples, while ZC3H13 silencing inhibited GC cell proliferation, migration, and invasion. Silencing ZC3H13 also enhanced the inhibitory effects of sevoflurane on GC cell malignancy. Moreover, we found that the increased expression of DLX6-AS1 in GC cells could be suppressed by ZC3H13 through the mediation of the m6A modification of DLX6-AS1, thereby reducing DLX6-AS1 stability. In conclusion, ZC3H13 knockdown enhances the inhibitory effect of sevoflurane on GC cell malignancy by inducing DLX6-AS1 m6A modification. Our findings may help identify potential therapeutic targets for the treatment of GC.

Search for Expression Marker Genes That Reflect the Physiological Conditions of Blossom End Enlargement Occurrence in Cucumber.

Blossom end enlargement (BEE) is a postharvest deformation that may be related to the influx of photosynthetic assimilates before harvest. To elucidate the mechanism by which BEE occurs, expression marker genes that indicate the physiological condition of BEE-symptomatic fruit are necessary. First, we discovered that preharvest treatment with a synthetic cytokinin, N-(2-Chloro-4-pyridyl)-N'-phenylurea (CPPU), promoted fruit growth and suppressed BEE occurrence. This suggests that excessive assimilate influx is not a main cause of BEE occurrence. Subsequently, the expression levels of seven sugar-starvation marker genes, CsSEF1, AS, CsFDI1, CsPID, CsFUL1, CsETR1, and CsERF1B, were compared among symptomatic and asymptomatic fruits, combined with and without CPPU treatment. Only CsSEF1 showed a higher expression level in asymptomatic fruits than in symptomatic fruits, regardless of CPPU treatment. This was then tested using fruits stored via the modified-atmosphere packaging technique, which resulted in a lower occurrence of BEE, and the asymptomatic fruits showed a higher CsSEF1 expression level than symptomatic fruits, regardless of the packaging method. CsSEF1 codes a CCCH-type zinc finger protein, and an increase in the expression of CsSEF1 was correlated with a decrease in the fruit respiration rate. Thus, CsSEF1 may be usable as a BEE expression marker gene.

Structure and RNA-binding of the helically extended Roquin CCCH-type zinc finger.

Zinc finger (ZnF) domains appear in a pool of structural contexts and despite their small size achieve varying target specificities, covering single-stranded and double-stranded DNA and RNA as well as proteins. Combined with other RNA-binding domains, ZnFs enhance affinity and specificity of RNA-binding proteins (RBPs). The ZnF-containing immunoregulatory RBP Roquin initiates mRNA decay, thereby controlling the adaptive immune system. Its unique ROQ domain shape-specifically recognizes stem-looped cis-elements in mRNA 3'-untranslated regions (UTR). The N-terminus of Roquin contains a RING domain for protein-protein interactions and a ZnF, which was suggested to play an essential role in RNA decay by Roquin. The ZnF domain boundaries, its RNA motif preference and its interplay with the ROQ domain have remained elusive, also driven by the lack of high-resolution data of the challenging protein. We provide the solution structure of the Roquin-1 ZnF and use an RBNS-NMR pipeline to show that the ZnF recognizes AU-rich RNAs. We systematically refine the contributions of adenines in a poly(U)-background to specific complex formation. With the simultaneous binding of ROQ and ZnF to a natural target transcript of Roquin, our study for the first time suggests how Roquin integrates RNA shape and sequence features through the ROQ-ZnF tandem.

The Stop Codon after the nsp3 Gene of Ross River Virus (RRV) Is Not Essential for Virus Replication in Three Cell Lines Tested, but RRV Replication Is Attenuated in HEK 293T Cells.

A recombinant Ross River virus (RRV) that contains the fluorescent protein mCherry fused to the non-structural protein 3 (nsP3) was constructed, which allowed real-time imaging of viral replication. RRV-mCherry contained either the natural opal stop codon after the nsP3 gene or was constructed without a stop codon. The mCherry fusion protein did not interfere with the viral life cycle and deletion of the stop codon did not change the replication capacity of RRV-mCherry. Comparison of RRV-mCherry and chikungunya virus-mCherry infections, however, showed a cell type-dependent delay in RRV-mCherry replication in HEK 293T cells. This delay was not caused by differences in cell entry, but rather by an impeded nsP expression caused by the RRV inhibitor ZAP (zinc finger CCCH-Type, antiviral 1). The data indicate that viral replication of alphaviruses is cell-type dependent, and might be unique for each alphavirus.

Multiomics Analyses Reveal MsC3H29 Positively Regulates Flavonoid Biosynthesis to Improve Drought Resistance of Autotetraploid Cultivated Alfalfa (Medicago sativa L.).

Alfalfa (Medicago sativa subsp. sativa), the "queen of forage," is the most important perennial legume, with high productivity and an excellent nutritional profile. Medicago sativa subsp. falcata is a subspecies of the alfalfa complex and exhibits better drought tolerance. However, drought stress significantly hampers their development and yield. The molecular mechanisms underlying the aboveground and underground tissues of sativa and falcata responding to drought stress remain obscure. Here, we performed a comprehensive comparative analysis of the physiological and transcriptomic responses of sativa and falcata under drought stress. The results showed that photosynthesis was inhibited, and antioxidant enzymes were activated under drought stress. MsC3H29, a CCCH-type zinc finger protein, was identified as a hub gene through weighted gene coexpression network analysis (WGCNA) and was significantly induced by drought in underground tissue. The MsC3H29 protein was localized in the nucleus. Overexpression (OE) of MsC3H29 can increase the primary root length and fresh weight of transgenic alfalfa hairy roots, while RNA interference (RNAi) decreases them under drought stress. The 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) staining revealed that MsC3H29 promoted drought tolerance of alfalfa hairy roots through decreasing ROS accumulation. The targeted metabolome analysis showed that the overexpression of MsC3H29 resulted in higher levels of accumulation for flavonoid monomers, including vicenin, daidzein, apigenin, isorhamnetin, quercetin, and tricin, in transgenic alfalfa hairy roots before and after drought stress, while RNAi led to a reduction. Our study provided a key candidate gene for molecular breeding to improve drought resistance in alfalfa.

Hsa_circ_0101050 regulated by ZC3H13 enhances tumorigenesis in papillary thyroid cancer via m6A modification

While the regulatory roles of circular RNAs (circRNAs) and zinc finger CCCH-type containing 13 (ZC3H13) were previously reported in various human cancers, the mechanisms underlying their interaction in papillary thyroid cancer (PTC) remain unclear. We aimed to determine the role of hsa_circ_0101050 and its regulatory relationship with ZC3H13 in PTC. The expression levels of hsa_circ_0101050 and ZC3H13 were determined in tumor samples and adjacent normal tissues from 46 patients with PTC and in two PTC cell lines (IHH-4 and PTC-1) using quantitative reverse transcription–polymerase chain reaction. The roles of hsa_circ_0101050 and ZC3H13 in cell viability, wound healing, and migration were determined using knockdown and overexpression approaches in PTC cell lines, and a xenograft model in nude mice was used to determine their role in vivo. Methylated RNA immunoprecipitation assay was used to analyze N6-methyladenosine (m6A) modification of hsa_circ_0101050 by ZC3H13. We found hsa_circ_0101050 overexpression and ZC3H13 downregulation in PTC samples and PTC cell lines. In PTC cell lines, silencing hsa_circ_0101050 reduced cell viability and migration whereas its overexpression promoted an aggressive PTC phenotype. ZC3H13 increased the m6A modification of hsa_circ_0101050 and repressed its expression. ZC3H13 overexpression inhibited PTC cell viability, migration, and invasion, which were reversed in cells overexpressing hsa_circ_0101050. Taken together, these results suggested that the downregulation of hsa_circ_0101050 mediated by ZC3H13 through m6A modification contributed to its oncogenic effect in PTC development, revealing the ZC3H13–m6A–hsa_circ_0101050 as a potential therapeutic target in PTC.

A nanopore-based cucumber genome assembly reveals structural variations at two QTLs controlling hypocotyl elongation.

The Xishuangbanna (XIS) cucumber (Cucumis sativus var. xishuangbannanesis) is a semiwild variety that has many distinct agronomic traits. Here, long reads generated by Nanopore sequencing technology helped assembling a high-quality genome (contig N50 = 8.7 Mb) of landrace XIS49. A total of 10,036 structural/sequence variations (SVs) were identified when comparing with Chinese Long (CL), and known SVs controlling spines, tubercles, and carpel number were confirmed in XIS49 genome. Two QTLs of hypocotyl elongation under low light, SH3.1 and SH6.1, were fine-mapped using introgression lines (donor parent, XIS49; recurrent parent, CL). SH3.1 encodes a red-light receptor Phytochrome B (PhyB, CsaV3_3G015190). A ∼4 kb region with large deletion and highly divergent regions (HDRs) were identified in the promoter of the PhyB gene in XIS49. Loss of function of this PhyB caused a super-long hypocotyl phenotype. SH6.1 encodes a CCCH-type zinc finger protein FRIGIDA-ESSENTIAL LIKE (FEL, CsaV3_6G050300). FEL negatively regulated hypocotyl elongation but it was transcriptionally suppressed by long terminal repeats retrotransposon insertion in CL cucumber. Mechanistically, FEL physically binds to the promoter of CONSTITUTIVE PHOTOMORPHOGENIC 1a (COP1a), regulating the expression of COP1a and the downstream hypocotyl elongation. These above results demonstrate the genetic mechanism of cucumber hypocotyl elongation under low light.

WITHDRAWN: Exploring the Interplay of m6A regulators in renal cell carcinoma: Molecular insights and clinical implications

BackgroundThe m6A modification of N6-methyladenosine (m6A) modification regulates gene expression and functioning by affecting RNA splicing, stability, translocation, and translation. ObjectiveThis study used comprehensive bioinformatics analysis to elucidate the mechanisms by which m6A regulators function in kidney renal clear cell carcinoma (KIRC) and their interrelationships. MethodsThe KIRC data extracted from The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) databases was used in this study. LASSO Cox regression, protein–protein interaction analysis, univariate Cox regression, and a predictive risk model. Cox regression, Kaplan–Meier analysis, consensus clustering, CIBERSORT, gene set enrichment analysis (GSEA), ESTIMATE, immune cell infiltration, and prognostic factors were investigated in KIRC utilizing computational methods. ResultsThe results of integrated analysis showed that KIRC has unique and complex gene patterns associated with m6A and is linked to different gene patterns. These patterns included an 11.68-fold change in insulin-like growth factor-2 mRNA-binding protein 3 (IGF2BP3), a 3.35-fold change in ELAV-like RNA binding protein 2 (ELAVL2), a 2.34-fold change in fat mass- and obesity-associated protein (FTO), a 2.52-fold change in ELAVL4, and a 2.15-fold change in ELAVL3. By contrast, there was downregulation of IGF2BP2, zinc finger CCCH-type containing 14 (ZC3H14), and alpha-ketoglutarate-dependent dioxygenase (ALKBH6), with fold changes of 0.44, 0.66, and 0.68, respectively. Minor modifications ranging from 1 to 1.28 were seen in other m6A regulators. ConclusionThis study showed that m6A alteration affects KIRC tumor immunity. Moreover, m6A regulators such as IGF2BP3 have therapeutic and diagnostic potential, which could improve patient outcomes by personalized immunotherapy.

Identification of differentially methylated regions associated with both liver fibrosis and hepatocellular carcinoma

BackgroundLiver fibrosis is a major risk factor for hepatocellular carcinoma (HCC). We have previously reported that differentially methylated regions (DMRs) are correlated with the fibrosis stages of metabolic dysfunction-associated steatotic liver disease (MASLD). In this study, the methylation levels of those DMRs in liver fibrosis and subsequent HCC were examined.MethodsThe methylation levels of DMRs were investigated using alcoholic cirrhosis and HCC (GSE60753). The data of hepatitis C virus-infected cirrhosis and HCC (GSE60753), and two datasets (GSE56588 and GSE89852) were used for replication analyses. The transcriptional analyses were performed using GSE114564, GSE94660, and GSE142530.ResultsHypomethylated DMR and increased transcriptional level of zinc finger and BTB domain containing 38 (ZBTB38) were observed in HCC. Hypermethylated DMRs, and increased transcriptional levels of forkhead box K1 (FOXK1) and zinc finger CCCH-type containing 3 (ZC3H3) were observed in HCC. The methylation levels of DMR of kazrin, periplakin interacting protein (KAZN) and its expression levels were gradually decreased as cirrhosis progressed to HCC.ConclusionsChanges in the methylation and transcriptional levels of ZBTB38, ZC3H3, FOXK1, and KAZN are important for the development of fibrosis and HCC; and are therefore potential therapeutic targets and diagnostic tools for cirrhosis and HCC.

ZC3H12A inhibits tumor growth and metastasis of breast cancer under hypoxic condition via the inactivation of IL-17 signaling pathway

ABSTRACT Hypoxia is a major contributor to tumor microenvironment (TME) and metastasis in most solid tumors. We seek to screen hypoxia-related genes affecting metastasis in breast cancer and to reveal relative potential regulatory pathway. Based on gene expression profiling of GSE17188 dataset, differential expressed genes (DEGs) were identified between highly metastatic breast cancer cells under hypoxia and samples under normoxia. The protein–protein interaction (PPI) network was utilized to determine hub genes. The gene expression profiling interactive analysis database (GEPIA2) and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) were employed to quantify hub genes. Moreover, overexpression of zinc finger CCCH-type containing 12A (ZC3H12A) was performed both in breast cancer cells and xenograft mouse model to determine the role of ZC3H12A. We identified 134 DEGs between hypoxic and normoxic samples. Based on PPI analysis, 5 hub genes interleukin (IL)-6, GALN (GAL), CD22 molecule (CD22), ZC3H12A and TNF receptor associated factor 1 (TRAF1) were determined; the expression levels of TRAF1, IL-6, ZC3H12A and GAL were remarkably downregulated while CD22 was upregulated in breast cancer cells. Besides, patients with higher expression of ZC3H12A had favorable prognosis. Overexpression of ZC3H12A could inhibit metastasis and tumor growth of breast cancer; overexpression of ZC3H12A downregulated the expression of IL-17 signaling pathway-related proteins such as IL-17 receptor A (IL-17RA), IL-17A and nuclear factor κB activator 1 (Act1). This study reveals ZC3H12A and IL-17 signaling pathway as potential therapeutic targets for hypoxic breast cancer.

Genome-wide identification, characterization, and expression analysis of m6A readers-YTH domain-containing genes in alfalfa

Eukaryotic messenger RNAs (mRNAs) are often modified with methyl groups at the N6 position of adenosine (m6A), and these changes are interpreted by YTH domain-containing proteins to regulate the metabolism of m6A-modified mRNAs. Although alfalfa (Medicago sativa) is an established model organism for forage development, the understanding of YTH proteins in alfalfa is still limited. In the present investigation, 53 putative YTH genes, each encoding a YT521 domain-containing protein, were identified within the alfalfa genome. These genes were categorized into two subfamilies: YTHDF (49 members) and YTHDC (four members). Each subfamily demonstrates analogous motif distributions and domain architectures. Specifically, proteins encoded by MsYTHDF genes incorporate a single domain structure, while those corresponding to MsYTH5, 8, 12, 16 who are identified as members of the MsYTHDC subfamily, exhibit CCCH-type zinc finger repeats at their N-termini. It is also observed that the predicted aromatic cage pocket that binds the m6A residue of MsYTHDC consists of a sequence of two tryptophan residues and one tyrosine residue (WWY). Conversely, in MsYTHDF, the binding pocket comprises two highly conserved tryptophan residues and either one tryptophan residue (WWW) or tyrosine residue (WWY) in MsYTHDF.Through comparative analysis of qRT-PCR data, we observed distinct expression patterns in specific genes under abiotic stress, indicating their potential regulatory roles. Notably, five genes (MsYTH2, 14, 26, 27, 48) consistently exhibit upregulation, and two genes (MsYTH33, 35) are downregulated in response to both cold and salt stress. This suggests a common mechanism among these YTH proteins in response to various abiotic stressors in alfalfa. Further, integrating qRT-PCR with RNA-seq data revealed that MsYTH2, MsYTH14, and MsYTH16 are highly expressed in leaves at various development stages, underscoring their potential roles in regulating the growth of these plant parts. The obtained findings shed further light on the biological functions of MsYTH genes and may aid in the selection of suitable candidate genes for future genetic enhancement endeavors aimed at improving salt and cold tolerance in alfalfa.

Cytokine modification by the RNA regulatory protein TFL

We found a novel gene, named "transformed follicular lymphoma" (TFL), in a patient who developed diffuse large B-cell lymphoma from follicular lymphoma. TFL modulates several cytokines and chemokines by binding their 3'UTR region of mRNA with its unique RNase motif. TFL is part of a family called zinc finger CCCH-type containing 12A-D. Due to its unique RNase motif, the TFL family is also called regulatory RNase (Regnase 1-4). TFL is expressed in lymphoid tissue and is upregulated by an inflammatory response, contributing to autoimmune diseases such as multiple sclerosis via IL-17 in CNS and worsening inflammation (cachexia) due to lymphoma progression through CXCL-13. Loss of TFL expression is reported to be a valuable biomarker for various cancers, including lung adenocarcinoma, endometrial cancer, and possibly lymphoma. In addition to its mRNA modulation function, Regnase1 is known to have deubiquitinase activity for TRAF2, 3, and 6, attenuating JNK and NFκB activity, and TFL captures and transports naked nonvesicular extracellular mRNA of IL-1β to the nucleus, enhancing the tumor-killing activity in NK cells. Based on its potential to modulate inflammation, TFL could be a future treatment target for autoimmune diseases and cancer.

Antiviral Activity of Zinc Finger Antiviral Protein (ZAP) in Different Virus Families.

The CCCH-type zinc finger antiviral protein (ZAP) in humans, specifically isoforms ZAP-L and ZAP-S, is a crucial component of the cell's intrinsic immune response. ZAP acts as a post-transcriptional RNA restriction factor, exhibiting its activity during infections caused by retroviruses and alphaviruses. Its function involves binding to CpG (cytosine-phosphate-guanine) dinucleotide sequences present in viral RNA, thereby directing it towards degradation. Since vertebrate cells have a suppressed frequency of CpG dinucleotides, ZAP is capable of distinguishing foreign genetic elements. The expression of ZAP leads to the reduction of viral replication and impedes the assembly of new virus particles. However, the specific mechanisms underlying these effects have yet to be fully understood. Several questions regarding ZAP's mechanism of action remain unanswered, including the impact of CpG dinucleotide quantity on ZAP's activity, whether this sequence is solely required for the binding between ZAP and viral RNA, and whether the recruitment of cofactors is dependent on cell type, among others. This review aims to integrate the findings from studies that elucidate ZAP's antiviral role in various viral infections, discuss gaps that need to be filled through further studies, and shed light on new potential targets for therapeutic intervention.

Non-Tandem CCCH-Type Zinc-Finger Protein CpZF_CCCH1 Improves Fatty Acid Desaturation and Stress Tolerance in Chlamydomonas reinhardtii.

The properties and nutritional value of microalgal bioproducts depend significantly on fatty acid desaturation, which is generally modulated by manipulating the culture conditions or associated gene expressions. Here, we investigated the role of CpZF_CCCH1, a non-tandem CCCH-type zinc-finger (non-TZF) protein, in elevating polyunsaturated fatty acid (PUFA) content (11.00-16.36%) in Chlamydomonas reinhardtii. Through lipidomic and flow cytometry analyses, we observed reduced triacylglycerol accumulation (7.01-21.15%) and elevated levels of membrane lipids containing PUFAs (7.81-46.18%) in C. reinhardtii overexpressing CpZF_CCCH1. Additionally, overexpression of nucleus-located CpZF_CCCH1 downregulated genes associated with triacylglycerol assembly and lipid turnover from 2.00- to 2.90-fold, likely by binding to GCN4 motif and promoter of 3-phosphate-glycerol acyltransferase. Furthermore, overexpression of CpZF_CCCH1 alleviated reactive oxygen species levels by 59.28-73.26% and enhanced stress tolerance under adverse conditions. These findings expanded the roles of non-TZF proteins in lipid metabolism, opening new avenues for metabolic engineering to enhance the nutritional value and stress tolerance of microalgae and agricultural crops.

Hypoxia-Induced Neuronal Activity in Glioma Patients Polarizes Microglia by Potentiating RNA m6A Demethylation.

Neuronal activity in the brain has been reported to promote the malignant progression of glioma cells via nonsynaptic paracrine and electrical synaptic integration mechanisms. However, the interaction between neuronal activity and the immune microenvironment in glioblastoma (GBM) remains largely unclear. By applying chemogenetic techniques, we enhanced and inhibited neuronal activity in vitro and in a mouse model to study how neuronal activity regulates microglial polarization and affects GBM progression. We demonstrate that hypoxia drove glioma stem cells (GSC) to produce higher levels of glutamate, which activated local neurons. Neuronal activity promoted GBM progression by facilitating microglial M2 polarization through enriching miR-200c-3p in neuron-derived exosomes, which decreased the expression of the m6A writer zinc finger CCCH-type containing 13 (ZC3H13) in microglia, impairing methylation of dual specificity phosphatase 9 (DUSP9) mRNA. Downregulation of DUSP9 promoted ERK pathway activation, which subsequently induced microglial M2 polarization. In the mouse model, cortical neuronal activation promoted microglial M2 polarization whereas cortical neuronal inhibition decreased microglial M2 polarization in GBM xenografts. miR-200c-3p knockdown in cortical neurons impaired microglial M2 polarization and GBM xenograft growth, even when cortical neurons were activated. Treatment with the anti-seizure medication levetiracetam impaired neuronal activation and subsequently reduced neuron-mediated microglial M2 polarization. These findings indicated that hypoxic GSC-induced neuron activation promotes GBM progression by polarizing microglia via the exosomal miR-200c-3p/ZC3H13/DUSP9/p-ERK pathway. Levetiracetam, an antiepileptic drug, blocks the abnormal activation of neurons in GBM and impairs activity-dependent GBM progression. See related commentary by Cui et al., p. 1073.

Characterization and Expression Analysis of HbC3H66: Implications for Transcriptional Regulation in Rubber Biosynthesis and Abiotic Stress Responses in Hevea brasiliensis

CCCH-type zinc-finger protein (ZnF_CCCH) family is one of the most important transcription factors (TFs) linked to various biotic and abiotic stressors and physiological and developmental processes in plants. This study aims to clone and characterise the latex gene expression patterns, evolution and characteristics of the Hevea brasiliensis ZnF_CCCH domain-containing protein 66 (HbC3H66) gene. These results showed that the open reading frame (ORF) of the HbC3H66 gene was 2,106 bp, encoding 701 aa and the calculated molecular weight of the encoded protein was 76.52 kDa. The N-terminal region of HbC3H66 contains 2 ANK repeats in, PfK_2/FBPase-2 and 2 type zinc finger motifs. Phylogenetic analysis showed that the C3H66 amino acid from Hevea and other plant C3H66 were clustered into 1 group and could be used for evolutionary analysis. Semi-quantitative RT-PCR (sqRT-PCR) revealed that the HbC3H66 mRNA was abundance in high-yielding Hevea clones (RRIT251 and RRIM600) and treatment with 2.5 % of ethephon (Eth) induced HbC3H66 mRNA expression in the latex of 15 years old Hevea trees. The HbC3H66 gene was induced by water deficit and 0.5 M NaCl in the latex of 3-month-old Hevea. In conclusion, the HbC3H66 protein may play a role in DNA-binding transcriptional regulation in NR pathways. Which would provide a theoretical basis for understanding the evolution and functions of the Hevea C3H66 gene in rubber biosynthesis. HIGHLIGHTS CCCH-type zinc-finger proteins (ZnF_CCCH) are essential transcription factors (TFs) for protein binding activity in plant growth and stress tolerance. Hevea brasiliensis zinc finger CCCH domain-containing protein 66 (HbC3H66) gene was clone and characterized. HbC3H66 contains 2 ANK repeat in, Pfk_2/FBPase-2 and 2 type zinc finger motifs. Semi-quantitative RT-PCR revealed that HbC3H66 gene was abundance in high-yielding Hevea clones (RRIT251 and RRIM600) and ethylene was induced HbC3H66 mRNA expression in the latex of Hevea The HbC3H66 gene was induced by water deficit and 0.5 M NaCl in the latex of seedling of Hevea trees. The HbC3H66 protein play a role in DNA-binding transcriptional regulation in NR pathway. GRAPHICAL ABSTRACT

Identification of CCCH-type zinc finger antiviral protein 1 (ZAP) gene from Pacific white shrimp (Penaeus vannamei): Characterization and expression analysis in response to viral infection.

Zinc-finger proteins (ZFPs) are a huge family that exert multiple roles in the cells. ZFPs could be divided into nine types based on the numbers and positions of conserved Cys and His residues, in which CCCH-type ZFP was one of the most widely studied types. CCCH-type zinc finger antiviral protein 1 (ZAP), a CCCH-type ZFP that can inhibit the replication of certain RNA viruses and DNA viruses by mediating degradation of viral RNA and repressing mRNA translation, plays significant roles in the host innate immune defenses against viral infections. Presently, there have been numerous reports investigating the antiviral ability of ZAP, while no data is available about ZAP gene in the species of shrimps or even crustaceans. In this study, a novel protein containing CCCH-type zinc finger motifs (ZnF-CCCH), CCCH-type zinc finger antiviral protein 1 (ZAP) gene, was identified from Pacific white shrimp (Penaeus vannamei) and its role in antiviral immunity was further investigated. Similar to mammalian ZAPs, in addition to ZnF-CCCH, PvZAP also possesses central WWE domains and C-terminal PARP domain. Phylogenetic analysis showed that PvZAP was close to that of the crustacean Pacific oyster, separating from the cluster of vertebrate ZAP proteins. Upon in vivo infection by IHHNV, gene expression of PvZAP was strongly up-regulated in the hepatopancreas and gills of both adult and juvenile shrimps, where adult individuals showed higher fold changes of up-regulation than in juvenile individuals. These results suggested that PvZAP might play an important role in the innate immune defense of Pacific white shrimp against IHHNV infection. This allows us to gain new insights into the immunological function of ZAP in the innate immunity of shrimp species and even crustaceans.

Expression of candidate marker genes of sugar starvation is upregulated in growth-suppressed parthenocarpic cucumber fruit. Novel gene markers for sugar starvation in growth-suppressed cucumber fruit.

To examine the physiological change in the growth suppression and abortion of parthenocarpic cucumber fruit, the expression of candidate marker genes of sugar starvation in relation to growth activity was examined. Fruits that failed to start exponential growth seemed to eventually abort. Hexose concentration of fruits was low in growth-suppressed fruit and increased in normally growing fruit consistent with the vacuolization. The correlation matrix indicated that the transcript levels of the genes, except CsaV3_6G046050 and CsaV3_5G032930, had a highly significant negative correlation with the relative growth rate in fruit length and had highly significant mutual positive correlations, suggesting that the asparagine synthetase gene, Cucumis sativus putative CCCH-type zinc finger protein CsSEF1, C. sativus BTB/POZ domain-containing protein At1g63850-like, CsaV3_3G000800, CsaV3_3G041280, and CsaV3_7G032930 are good markers of sugar starvation in cucumber fruit. The expression of candidate marker genes together with the hexose analysis strongly suggests that severe sugar starvation is occurring in growth-suppressed fruit.