CCCH-type Zinc Finger Research Articles

Data-independent acquisition-based global phosphoproteomics reveal the diverse roles of casein kinase 1 in plant development

Casein kinase 1 (CK1) is serine/threonine protein kinase highly conserved among eukaryotes, and regulates multiple developmental and signaling events through phosphorylation of target proteins. Arabidopsis early flowering 1 (EL1)-like (AELs) are plant-specific CK1s with varied functions, but identification and validation of their substrates is a major bottleneck in elucidating their physiological roles. Here, we conducted a quantitative phosphoproteomic analysis in data-independent acquisition mode to systematically identify CK1 substrates. We extracted proteins from seedlings overexpressing individual AEL genes (AEL1/2/3/4-OE) or lacking AEL function (all ael single mutants and two triple mutants) to identify the high-confidence phosphopeptides with significantly altered abundance compared to wild-type Col-0. Among these, we selected 3985 phosphopeptides with higher abundance in AEL-OE lines or lower abundance in ael mutants compared with Col-0 as AEL-upregulated phosphopeptides, and defined 1032 phosphoproteins. Eight CK1s substrate motifs were enriched among AEL-upregulated phosphopeptides and verified, which allowed us to predict additional candidate substrates and functions of CK1s. We functionally characterized a newly identified substrate C3H17, a CCCH-type zinc finger transcription factor, through biochemical and genetic analyses, revealing a role for AEL-promoted C3H17 protein stability and transactivation activity in regulating embryogenesis. As CK1s are highly conserved across eukaryotes, we searched the rice, mouse, and human protein databases using newly identified CK1 substrate motifs, yielding many more candidate substrates than currently known, largely expanding our understanding of the common and distinct functions exerted by CK1s in Arabidopsis and humans, facilitating future mechanistic studies of CK1-mediated phosphorylation in different species.

Masculinizer gene controls sexual differentiation in Hyphantria cunea.

The Masculinizer gene, Masc, encodes a lepidopteran-specific novel CCCH-type zinc finger protein, which controls sex determination and dosage compensation in Bombyx mori. Considering the potential application of it in pest control, it is necessary to investigate the function of Masc gene in Hyphantria cunea, a globally invasive forest pest. In the present study, we identified and functionally characterized the Masc gene, HcMasc, in H. cunea. Sequence analysis revealed that HcMASC contained the conserved CCCH-type zinc finger domain, nuclear localization signal, and male determining domain, in which the last was confirmed to be required for its masculinization in BmN cell line. However, expression data showed that unlike male-biased expression in B. mori, HcMasc gene expresses in main all developmental stages or tissues in both sexes. Clustered regularly interspaced palindromic repeats (CRISPR) / CRISPR-associated protein 9-based disruption of the common exons 1 and 3 of the HcMasc gene resulted in imbalanced sex ratio and abnormal external genitalia of both sexes. Our results suggest that the HcMasc gene is required for both male and female sexual differentiation and dosage compensation in H. cunea and provide a foundation for developing better strategies to control this pest.

ZCCHC3 is a stress granule zinc knuckle protein that strongly suppresses LINE-1 retrotransposition.

Retrotransposons have generated about half of the human genome and LINE-1s (L1s) are the only autonomously active retrotransposons. The cell has evolved an arsenal of defense mechanisms to protect against retrotransposition with factors we are only beginning to understand. In this study, we investigate Zinc Finger CCHC-Type Containing 3 (ZCCHC3), a gag-like zinc knuckle protein recently reported to function in the innate immune response to infecting viruses. We show that ZCCHC3 also severely restricts human retrotransposons and associates with the L1 ORF1p ribonucleoprotein particle. We identify ZCCHC3 as a bona fide stress granule protein, and its association with LINE-1 is further supported by colocalization with L1 ORF1 protein in stress granules, dense cytoplasmic aggregations of proteins and RNAs that contain stalled translation pre-initiation complexes and form when the cell is under stress. Our work also draws links between ZCCHC3 and the anti-viral and retrotransposon restriction factors Mov10 RISC Complex RNA Helicase (MOV10) and Zinc Finger CCCH-Type, Antiviral 1 (ZC3HAV1, also called ZAP). Furthermore, collective evidence from subcellular localization, co-immunoprecipitation, and velocity gradient centrifugation connects ZCCHC3 with the RNA exosome, a multi-subunit ribonuclease complex capable of degrading various species of RNA molecules and that has previously been linked with retrotransposon control.

Association of ZC3HAV1 single nucleotide polymorphisms with the susceptibility of Vogt-Koyanagi-Harada Disease

BackgroundPolymorphisms of genes related to the immune response have been reported to confer susceptibility to Vogt-Koyanagi-Harada (VKH) disease. This study was carried out to determine whether zinc finger CCCH-type containing antiviral 1 (ZC3HAV1) and tripartite motif-containing protein 25 (TRIM25) genetic polymorphisms are associated with this disease.MethodsA total of 766 VKH patients and 909 healthy individuals were enrolled in this two-stage case-control study. Thirty-one tag single nucleotide polymorphisms (SNPs) of ZC3HAV1 and TRIM25 were genotyped by MassARRAY System and iPLEX Gold Genotyping Assay. Allele and genotype frequencies were analyzed by the χ2 test or Fisher’s exact test. Cochran-Mantel-Haenszel test was used to assess the pooled odds ratio (OR) in the combined study. A stratified analysis was performed in terms of the major clinical features of VKH disease.ResultsWe found a statistically significant increased frequency of the minor A allele of ZC3HAV1 rs7779972 (P = 1.50 × 10− 4, pooled OR = 1.332, 95%CI = 1.149–1.545) in VKH disease as compared with controls by using the Cochran-Mantel-Haenszel test. The GG genotype of rs7779972 showed a protective association with VKH disease (P = 1.88 × 10− 3, OR = 0.733, 95%CI = 0.602–0.892). There was no difference regarding the frequency of the remaining SNPs between VKH cases and controls (all P > 2.08 × 10− 3). The stratified analysis showed no significant association of rs7779972 with the major clinical characteristics of VKH disease.ConclusionOur study indicated that the ZC3HAV1 variant rs7779972 might confer susceptibility to VKH disease in Han Chinese.

A positive feedback loop controls Toxoplasma chronic differentiation.

Successful infection strategies must balance pathogen amplification and persistence. In the obligate intracellular parasite Toxoplasma gondii this is accomplished through differentiation into dedicated cyst-forming chronic stages that avoid clearance by the host immune system. The transcription factor BFD1 is both necessary and sufficient for stage conversion; however, its regulation is not understood. In this study we examine five factors that are transcriptionally activated by BFD1. One of these is a cytosolic RNA-binding protein of the CCCH-type zinc-finger family, which we name bradyzoite formation deficient 2 (BFD2). Parasites lacking BFD2 fail to induce BFD1 and are consequently unable to fully differentiate in culture or in mice. BFD2 interacts with the BFD1 transcript under stress, and deletion of BFD2 reduces BFD1 protein levels but not messenger RNA abundance. The reciprocal effects on BFD2 transcription and BFD1 translation outline a positive feedback loop that enforces the chronic-stage gene-expression programme. Thus, our findings help explain how parasites both initiate and commit to chronic differentiation. This work provides new mechanistic insight into the regulation of T. gondii persistence, and can be exploited in the design of strategies to prevent and treat these key reservoirs of human infection.

Genomic Identification of CCCH-Type Zinc Finger Protein Genes Reveals the Role of HuTZF3 in Tolerance of Heat and Salt Stress of Pitaya (Hylocereus polyrhizus)

Pitaya (Hylocereus polyrhizus) is cultivated in a broad ecological range, due to its tolerance to drought, heat, and poor soil. The zinc finger proteins regulate gene expression at the transcriptional and post-transcriptional levels, by interacting with DNA, RNA, and proteins, to play roles in plant growth and development, and stress response. Here, a total of 81 CCCH-type zinc finger protein genes were identified from the pitaya genome. Transcriptomic analysis showed that nine of them, including HuTZF3, responded to both salt and heat stress. RT-qPCR results showed that HuTZF3 is expressed in all tested organs of pitaya, with a high level in the roots and stems, and confirmed that expression of HuTZF3 is induced by salt and heat stress. Subcellular localization showed that HuTZF3 is targeted in the processing bodies (PBs) and stress granules (SGs). Heterologous expression of HuTZF3 could improve both salt and heat tolerance in Arabidopsis, reduce oxidative stress, and improve the activity of catalase and peroxidase. Therefore, HuTZF3 may be involved in post-transcriptional regulation via localizing to PBs and SGs, contributing to both salt and heat tolerance in pitaya.

The CCCH-Type Zinc-Finger Protein GhC3H20 Enhances Salt Stress Tolerance in Arabidopsis thaliana and Cotton through ABA Signal Transduction Pathway

The CCCH zinc-finger protein contains a typical C3H-type motif widely existing in plants, and it plays an important role in plant growth, development, and stress responses. In this study, a CCCH zinc-finger gene, GhC3H20, was isolated and thoroughly characterized to regulate salt stress in cotton and Arabidopsis. The expression of GhC3H20 was up-regulated under salt, drought, and ABA treatments. GUS activity was detected in the root, stem, leaves, and flowers of ProGhC3H20::GUS transgenic Arabidopsis. Compared with the control, the GUS activity of ProGhC3H20::GUS transgenic Arabidopsis seedlings under NaCl treatment was stronger. Through the genetic transformation of Arabidopsis, three transgenic lines of 35S-GhC3H20 were obtained. Under NaCl and mannitol treatments, the roots of the transgenic lines were significantly longer than those of the wild-type (WT) Arabidopsis. The leaves of the WT turned yellow and wilted under high-concentration salt treatment at the seedling stage, while the leaves of the transgenic Arabidopsis lines did not. Further investigation showed that compared with the WT, the content of catalase (CAT) in the leaves of the transgenic lines was significantly higher. Therefore, compared with the WT, overexpression of GhC3H20 enhanced the salt stress tolerance of transgenic Arabidopsis. A virus-induced gene silencing (VIGS) experiment showed that compared with the control, the leaves of pYL156-GhC3H20 plants were wilted and dehydrated. The content of chlorophyll in pYL156-GhC3H20 leaves was significantly lower than those of the control. Therefore, silencing of GhC3H20 reduced salt stress tolerance in cotton. Two interacting proteins (GhPP2CA and GhHAB1) of GhC3H20 have been identified through a yeast two-hybrid assay. The expression levels of PP2CA and HAB1 in transgenic Arabidopsis were higher than those in the WT, and pYL156-GhC3H20 had expression levels lower than those in the control. GhPP2CA and GhHAB1 are the key genes involved in the ABA signaling pathway. Taken together, our findings demonstrate that GhC3H20 may interact with GhPP2CA and GhHAB1 to participate in the ABA signaling pathway to enhance salt stress tolerance in cotton.

Molecular Mapping of Biofortification Traits in Bread Wheat (Triticum aestivum L.) Using a High-Density SNP Based Linkage Map.

A set of 188 recombinant inbred lines (RILs) derived from a cross between a high-yielding Indian bread wheat cultivar HD2932 and a synthetic hexaploid wheat (SHW) Synthetic 46 derived from tetraploid Triticum turgidum (AA, BB 2n = 28) and diploid Triticum tauschii (DD, 2n = 14) was used to identify novel genomic regions associated in the expression of grain iron concentration (GFeC), grain zinc concentration (GZnC), grain protein content (GPC) and thousand kernel weight (TKW). The RIL population was genotyped using SNPs from 35K Axiom® Wheat Breeder's Array and 34 SSRs and phenotyped in two environments. A total of nine QTLs including five for GPC (QGpc.iari_1B, QGpc.iari_4A, QGpc.iari_4B, QGpc.iari_5D, and QGpc.iari_6B), two for GFeC (QGfec.iari_5B and QGfec.iari_6B), and one each for GZnC (QGznc.iari_7A) and TKW (QTkw.iari_4B) were identified. A total of two stable and co-localized QTLs (QGpc.iari_4B and QTkw.iari_4B) were identified on the 4B chromosome between the flanking region of Xgwm149-AX-94559916. In silico analysis revealed that the key putative candidate genes such as P-loop containing nucleoside triphosphatehydrolase, Nodulin-like protein, NAC domain, Purine permease, Zinc-binding ribosomal protein, Cytochrome P450, Protein phosphatase 2A, Zinc finger CCCH-type, and Kinesin motor domain were located within the identified QTL regions and these putative genes are involved in the regulation of iron homeostasis, zinc transportation, Fe, Zn, and protein remobilization to the developing grain, regulation of grain size and shape, and increased nitrogen use efficiency. The identified novel QTLs, particularly stable and co-localized QTLs are useful for subsequent use in marker-assisted selection (MAS).

ZC3H13 Enhances the Malignancy of Cervical Cancer by Regulating m6A Modification of CKAP2.

Sustained expression of zinc finger CCCH-type containing 13 (ZC3H13) in tumors is essential for cancer cell malignancy; however, our understanding of its clinical effects and mechanisms in cervical cancer (CC) is limited. In this study, we aimed to reveal the effect on CC progression of ZC3H13-mediated N6-methyladenosine (m6A) modification to stabilize cytoskeleton-associated protein 2 (CKAP2) expression. CC tissues and paired adjacent normal tissues were collected from 50 patients. qRT-PCR was used to clarify ZC3H13 and CKAP2 expression levels in the CC tissues. The functional roles of ZC3H13 and CKAP2 in CC were analyzed by detecting the changes in CC cell proliferation, migration, invasion, and tumor growth in vivo. The regulatory relationship between ZC3H13 and CKAP2 was investigated by confirming m6A modification levels and their expression correlation. ZC3H13 and CKAP2 were highly expressed in CC and linked with poor prognosis. We observed that ZC3H13 inhibition decreased CC cell proliferation, invasion, and migration, while its facilitation promoted CC cell malignancy. ZC3H13 mediated m6A modification of CKAP2 to enhance CKAP2 expression in CC cells. Furthermore, CKAP2 overexpression partially restored the malignant phenotypic promotion induced by ZC3H13 overexpression in CC cells. In summary, this study revealed that ZC3H13-mediating m6A modification of CKAP2 promotes CC development. This finding should be conducive to an understanding of the role of ZC3H13-m6A-CKAP2 in CC and should provide an effective therapeutic target for this cancer.

Cell-based analysis reveals that sex-determining gene signals in Ostrinia are pivotally changed by male-killing Wolbachia.

Wolbachia, a maternally transmitted bacterium, shows male-killing, an adaptive phenotype for cytoplasmic elements, in various arthropod species during the early developmental stages. In lepidopteran insects, lethality of males is accounted for by improper dosage compensation in sex-linked genes owing to Wolbachia-induced feminization. Herein, we established Ostrinia scapulalis cell lines that retained sex specificity per the splicing pattern of the sex-determining gene doublesex (Osdsx). We found that Wolbachia transinfection in male cell lines enhanced the female-specific splice variant of Osdsx (OsdsxF ) while suppressing the male-specific variant (OsdsxM ), indicating that Wolbachia affects sex-determining gene signals even in vitro. Comparative transcriptome analysis isolated only two genes that behave differently upon Wolbachia infection. The two genes were respectively homologous to Masculinizer (BmMasc) and zinc finger-2 (Bmznf-2), male-specifically expressed sex-determining genes of the silkworm Bombyx mori that encode CCCH-type zinc finger motif proteins. By using cultured cells and organismal samples, OsMasc and Osznf-2 were found to be sex-determining genes of O. scapulalis that are subjected to sex-specific alternative splicing depending upon the chromosomal sex, developmental stage, and infection status. Overall, our findings expound the cellular autonomy in insect sex determination and the mechanism through which sex is manipulated by intracellular selfish microbes.

OsTZF1, a CCCH-tandem zinc finger protein gene, driven under own promoter produces no pleiotropic effects and confers salt and drought tolerance in rice

ABSTRACT Different abiotic stresses induce OsTZF1, a tandem CCCH-type zinc finger domain gene, in rice. Here, we report that transgenic rice plants overexpressing OsTZF1 under own promoter (POsTZF1:OsTZF1-OX [for overexpression]) transferred to soil showed normal growth similar to vector control plants. The POsTZF1:OsTZF1-OX produced normal leaves without any lesion mimic phenotype and exhibited normal seed setting. The POsTZF1:OsTZF1-OX plants showed significantly increased tolerance to salt and drought stresses and enhanced post stress recovery. Microarray analysis revealed a total of 846 genes up-regulated and 360 genes down-regulated in POsTZF1:OsTZF1-OX salt-treated plants. Microarray analysis of POsTZF1:OsTZF1-OX plants showed the regulation of many abiotic stress tolerance genes. These results suggest that OsTZF1-OX under own promoter show abiotic stress tolerance and produces no pleiotropic effect on phenotype of transgenic rice plant.

A tandem CCCH type zinc finger protein gene CpC3H3 from Chimonanthus praecox promotes flowering and enhances drought tolerance in Arabidopsis

BackgroundCCCH-type zinc finger proteins play important roles in plant development and biotic/abiotic stress responses. Wintersweet (Chimonanthus praecox) is a popular ornamental plant with strong resistance to various stresses, which is a good material for exploring gene resource for stress response. In this study, we isolated a CCCH type zinc finger protein gene CpC3H3 (MZ964860) from flower of wintersweet and performed functional analysis with a purpose of identifying gene resource for floral transition and stress tolerance.ResultsCpC3H3 was predicted a CCCH type zinc finger protein gene encoding a protein containing 446 amino acids with five conserved C-X8-C-X5-C-X3-H motifs. CpC3H3 was localized in the cell membrane but with a nuclear export signal at the N-terminal. Transcripts of CpC3H3 were significantly accumulated in flower buds at floral meristem formation stage, and were induced by polyethylene glycol. Overexpression of CpC3H3 promoted flowering, and enhanced drought tolerance in transgenic A. thaliana. CpC3H3 overexpression affects the expression level of genes involved in flower inducement and stress responses. Further comparative studies on physiological indices showed the contents of proline and soluble sugar, activity of peroxidase and the rates of electrolyte leakage were significantly increased and the content of malondialdehyde and osmotic potential was significantly reduced in transgenic A. thaliana under PEG stress.ConclusionOverall, CpC3H3 plays a role in flowering inducement and drought tolerance in transgenic A. thaliana. The CpC3H3 gene has the potential to be used to promote flowering and enhance drought tolerance in plants.

Chrysanthemum MAF2 regulates flowering by repressing gibberellin biosynthesis in response to low temperature.

Chrysanthemum (Chrysanthemum morifolium) is well known as a photoperiod-sensitive flowering plant. However, it has also evolved into a temperature-sensitive ecotype. Low temperature can promote the floral transition of the temperature-sensitive ecotype, but little is known about the underlying molecular mechanisms. Here, we identified MADS AFFECTING FLOWERING 2 (CmMAF2), a putative MADS-box gene, which induces floral transition in response to low temperatures independent of day length conditions in this ecotype. CmMAF2 was shown to bind to the promoter of the GA biosynthesis gene CmGA20ox1 and to directly regulate the biosynthesis of bioactive GA1 and GA4 . The elevated bioactive GA levels activated LEAFY (CmLFY) expression, ultimately initiating floral transition. In addition, CmMAF2 expression in response to low temperatures was directly activated by CmC3H1, a CCCH-type zinc-finger protein upstream. In summary, our results reveal that the CmC3H1-CmMAF2 module regulates flowering time in response to low temperatures by regulating GA biosynthesis in the temperature-sensitive chrysanthemum ecotype.

Selection Signature and CRISPR/Cas9-Mediated Gene Knockout Analyses Reveal ZC3H10 Involved in Cold Adaptation in Chinese Native Cattle.

Cold stress is an important factor affecting cattle health, production performance, and reproductive efficiency. Understanding of the potential mechanism underlying genetic adaptation to local environments, particularly extreme cold environment, is limited. Here, by using FLK and hapFLK methods, we found that the Zinc finger CCCH-type containing 10 (ZC3H10) gene underwent positive selection in the Menggu, Fuzhou, Anxi, and Shigatse humped cattle breeds that are distributed in the cold areas of China. Furthermore, ZC3H10 expression significantly increased in bovine fetal fibroblast (BFF) cells at 28 °C for 4 h. ZC3H10 knockout BFFs were generated using CRISPR/Cas9. Wild and ZC3H10-deleted BFFs were treated at two temperatures and were divided into four groups (WT, wild and cultured at 38 °C; KO, ZC3H10−/− and 38 °C; WT_LT, wild, and 28 °C for 4 h; and KO_LT, ZC3H10−/− and 28 °C for 4 h. A total of 466, 598, 519, and 650 differently expressed genes (two-fold or more than two-fold changes) were identified by determining transcriptomic difference (KO_LT vs. KO, WT_LT vs. WT, KO vs. WT, and KO_LT vs. WT_LT, respectively). Loss of ZC3H10 dysregulated pathways involved in thermogenesis and immunity, and ZC3H10 participated in immunity-related pathways induced by cold stress and regulated genes involved in glucose and lipid metabolism and lipid transport (PLTP and APOA1), thereby facilitating adaptability to cold stress. Our findings provide a foundation for further studies on the function of ZC3H10 in cold stress and development of bovine breeding strategies for combatting the influences of cold climate.

CircZC3HAV1 Regulates TBC1D9 to Affect the Biological Behavior of Colorectal Cancer Cells.

Background Colorectal cancer (CRC) is one of the most frequently diagnosed cancers all over the world, which accounts for a large proportion of cancer-associated deaths. The regulatory function of circular RNAs (circRNAs) has been affirmed in diverse cancers. circ_0082628, named circRNA zinc finger CCCH-type containing antiviral 1 (circZC3HAV1), has been discovered to be significantly downregulated in CRC tissues. Nevertheless, the function and mechanism of circZC3HAV1 in CRC remain unclear. Purpose We targeted at studying the specific role and mechanism of circZC3HAV1 in CRC cells. Methods The expression of the genes was detected by quantitative real-time polymerase chain reaction (qPCR). The binding relationship among different genes was verified by mechanism assays. Functional assays were carried out to reveal the role of different RNAs in CRC cell malignant behaviors. Results circZC3HAV1 was significantly downregulated in CRC cells. circZC3HAV1 overexpression hampered CRC cell migratory and invasive abilities. As for the mechanism, circZC3HAV1 competitively bound with microRNA-146b-3p (miR-146b-3p) to enhance the expression of TBC1 domain family member 9 (TBC1D9). Rescue assays demonstrated circZC3HAV1 sponged miR-146b-3p and upregulated TBC1D9 to restrict migration and invasion of CRC cells. Conclusion circZC3HAV1 could upregulate TBC1D9 via absorbing miR-146b-3p, consequently inhibiting migratory and invasive capabilities of CRC cells.

Zrsr2 Is Essential for the Embryonic Development and Splicing of Minor Introns in RNA and Protein Processing Genes in Zebrafish.

ZRSR2 (zinc finger CCCH-type, RNA binding motif and serine/arginine rich 2) is an essential splicing factor involved in 3′ splice-site recognition as a component of both the major and minor spliceosomes that mediate the splicing of U2-type (major) and U12-type (minor) introns, respectively. Studies of ZRSR2-depleted cell lines and ZRSR2-mutated patient samples revealed its essential role in the U12-dependent minor spliceosome. However, the role of ZRSR2 during embryonic development is not clear, as its function is compensated for by Zrsr1 in mice. Here, we utilized the zebrafish model to investigate the role of zrsr2 during embryonic development. Using CRISPR/Cas9 technology, we generated a zrsr2-knockout zebrafish line, termed zrsr2hg129/hg129 (p.Trp167Argfs*9) and examined embryo development in the homozygous mutant embryos. zrsr2hg129/hg129 embryos displayed multiple developmental defects starting at 4 days post fertilization (dpf) and died after 8 dpf, suggesting that proper Zrsr2 function is required during embryonic development. The global transcriptome analysis of 3 dpf zrsr2hg129/hg129 embryos revealed that the loss of Zrsr2 results in the downregulation of essential metabolic pathways and the aberrant retention of minor introns in about one-third of all minor intron-containing genes in zebrafish. Overall, our study has demonstrated that the role of Zrsr2 as a component of the minor spliceosome is conserved and critical for proper embryonic development in zebrafish.

Transcriptome-Wide Identification of CCCH-Type Zinc Finger Proteins Family in Pinus massoniana and RR-TZF Proteins in Stress Response.

CCCH-type zinc finger proteins play an important role in multiple biotic and abiotic stresses. More and more reports about CCCH functions in plant development and stress responses have appeared over the past few years, focusing especially on tandem CCCH zinc finger proteins (TZFs). However, this has not been reported in Pinaceae. In this study, we identified 46 CCCH proteins, including 6 plant TZF members in Pinus massoniana, and performed bioinformatic analysis. According to RT-PCR analysis, we revealed the expression patterns of five RR-TZF genes under different abiotic stresses and hormone treatments. Meanwhile, tissue-specific expression analysis suggested that all genes were mainly expressed in needles. Additionally, RR-TZF genes showed transcriptional activation activity in yeast. The results in this study will be beneficial in improving the stress resistance of P. massoniana and facilitating further studies on the biological and molecular functions of CCCH zinc finger proteins.

STRONG STAYGREEN inhibits DNA binding of PvNAP transcription factors during leaf senescence in switchgrass.

Fine tuning the progression of leaf senescence is important for plant fitness in nature, while the "staygreen" phenotype with delayed leaf senescence has been considered a valuable agronomic trait in crop genetic improvement. In this study, a switchgrass (Panicum virgatum L.) CCCH-type Zinc finger gene, Strong Staygreen (PvSSG), was characterized as a suppressor of leaf senescence as overexpression or suppression of the gene led to delayed or accelerated leaf senescence, respectively. Transcriptomic analysis marked that chlorophyll (Chl) catabolic pathway genes were involved in the PvSSG-regulated leaf senescence. PvSSG was identified as a nucleus-localized protein with no transcriptional activity. By yeast two-hybrid screening, we identified its interacting proteins, including a pair of paralogous transcription factors, PvNAP1/2 (NAC-LIKE, ACTIVATED BY AP3/PI). Overexpression of PvNAPs led to precocious leaf senescence at least partially by directly targeting and transactivating Chl catabolic genes to promote Chl degradation. PvSSG, through protein-protein interaction, repressed the DNA-binding efficiency of PvNAPs and alleviated its transactivating effect on downstream genes, thereby functioning as a "brake" in the progression of leaf senescence. Moreover, overexpression of PvSSG resulted in up to 47% higher biomass yield and improved biomass feedstock quality, reiterating the importance of leaf senescence regulation in the genetic improvement of switchgrass and other feedstock crops.

AaZFP3, a Novel CCCH-Type Zinc Finger Protein from Adonis amurensis, Promotes Early Flowering in Arabidopsis by Regulating the Expression of Flowering-Related Genes.

CCCH-type zinc finger proteins (ZFP) are a large family of proteins that play various important roles in plant growth and development; however, the functions of most proteins in this family are uncharacterized. In this study, a CCCH-type ZFP, AaZFP3, was identified in the floral organ of Adonis amurensis. Quantitative real-time PCR (qPCR) analysis revealed that AaZFP3 was widely expressed in the flowers of A. amurensis. Subcellular localization analysis showed that the AaZFP3 protein was mainly localized to the cytoplasm in tobacco and Arabidopsis. Furthermore, the overexpression of AaZFP3 promoted early flowering in Arabidopsis under both normal and relatively low-temperature conditions. RNA-sequencing and qPCR analyses revealed that the expression of multiple key flowering-time genes was altered in transgenic Arabidopsis overexpressing AaZFP3 compared to wild-type. Of these genes, FLOWERING LOCUS T (AtFT) expression was most significantly up-regulated, whereas FLOWERING LOCUS C (AtFLC) was significantly down-regulated. These results suggest that the overexpression of AaZFP3 promotes early flowering in Arabidopsis by affecting the expression of flowering-time genes. Overall, our study indicates that AaZFP3 may be involved in flowering regulation in A. amurensis and may represent an important genetic resource for improving flowering-time control in other ornamental plants or crops.

Enterovirus 71 VP1 promotes 5-HT release by upregulating the expression of ERICH3 and methyltransferase ZC3H13

Background and aimThe effect of structural viral protein 1 (VP1) on neurological damage caused by enterovirus 71 (EV71) infection is unclear. This study aimed to explore the transcriptome changes in EV infected patients and the role of VP1 on the cell secretion pathway of neuron cells. MethodsIn our cohort, EV infected patients were enrolled, and RNA-seq analysis was used to evaluate the distinct transcript patterns of cerebrospinal fluid (CSF). The EV71 VP1-overexpressing vector (pEGFP-c3-VP1) was generated and transfected into neuron cells. The relationship between Glutamate Rich 3 (ERICH3) and methyltransferase Zinc Finger CCCH-Type Containing 13 (ZC3H13) and their effect on the serotonin (5-HT) release of neuron cells were explored using small interfering RNA. The expression of ERICH3 and ZC3H13 and concentration of 5-HT were determined using real-time PCR, Western blot, and ELISA, respectively. ResultThe expression of ERICH3 and ZC3H13 were significantly upregulated in EV infected patients with neurological symptoms compared to those without (P < 0.05). The ERICH3 gene had many N6-methyladenosine (m6A) binding sites that can be regulated by m6A modification. Further, the expression of ERICH3 and ZC3H13 were elevated significantly in EV71-VP1 overexpressing neuron cells (P < 0.05). Moreover, ERICH3 or ZC3H13 deficiency could significantly downregulate the release of 5-HT in VP1-overexpressing cells (P < 0.05). Nonetheless, ERICH3 expression was significantly suppressed when ZC3H13 was silenced in neuron cells and vice versa (P < 0.05). ConclusionsEV71-VP1 can promote 5-HT release by upregulating the expression of ERICH3 and ZC3H13. 5-HT might be a novel therapeutic target for EV71 infection-induced fatal neuronal damage.