Genome-wide identification and expression analysis of Gossypium RING-H2 finger E3 ligase genes revealed their roles in fiber development,and phytohormone and abiotic stress responses

Background： RING H2 finger E3 ligase （RH2FE3） genes encode cysteine rich proteins that mediate E3 ubiquitin ligase activity and degrade target substrates. The roles of these genes in plant responses to phytohormones and abiotic stresses are well documented in various species, but their roles in cotton fiber development are poorly understood. To date, genome wide identification and expression analyses of Gossypium hirsutum RH2FE3 genes have not been reported. Methods： We performed computational identification, structural and phylogenetic analyses, chromosomal distribution analysis and estimated KJKs values of G hirsutum RH2FE3 genes. Orthologous and paralogous gene pairs were identified by all versus all BLASTP searches. We predicted cis regulatory elements and analyzed microarray data sets to generate heatmaps at different development stages. Tissue specific expression in cotton fiber, and hormonal and abiotic stress responses were determined by quantitative real time polymerase chain reaction （qRT PCR） analysis. Results： We investigated 140 G hirsutum, 80 G. orboreum, and evolutionary mechanisms and compared them with orthologs 89 G. roimondii putative RH2FB genes and their in Arobidopsis and rice. A domain based analysis of the G hirsutum RH2FE3 genes predicted conserved signature motifs and gene structures. Chromosomal localization showed the genes were distributed across all G hirsutum chromosomes, and 60 duplication events （4 tandem and 56 segmental duplications） and 98 orthologs were detected, cis elements were detected in the promoter regions of G hirsutum RH2FE3 genes. Microarray data and qRT PCR analyses showed that G hirsutum RH2FE3 genes were strongly correlated with cotton fiber development. Additionally, almost all the （brassinolide, gibberellic acid （GA）, indole 3-acetic acid drought, and salt）. dentified genes were up regulated in response to phytohormones （IAA）, and salicylic acid （SA）） and abiotic stresses （cold, heat, Conclusions： The genome wide identification, comprehensive analysis, and characterization of conserved domains and gene structures, as well as phylogenetic analysis, cis element prediction, and expression profile analysis of G hirsutum RH2FE3 genes and their roles in cotton fiber development and responses to plant hormones and abiotic stresses are reported here for the first time. Our findings will contribute to the genome wide analysis of putative RH2FE3 genes in other species and lay a foundation for future physiological and functional research on G hirsutum RH2FE3 genes.

TaGW2L,a GW2-like RING finger E3 ligase,positively regulates heading date in common wheat(Triticum aestivum L.)

RING finger E3 ligases play an important role in regulating plant growth and development by mediating substrate degradation.In this study,we identified TaGW2L,encoding a Grain width and weight2(GW2)-like RING finger E3 ligase,as a novel positive regulator of heading date in wheat(Triticum aestivum L.).TaGW2L exhibited high amino acid sequence similarities with TaGW2 homoeologs,particularly in the conserved RING finger domain.Expression analysis indicated that TaGW2L was constitutively expressed in various wheat tissues.TaGW2L showed transactivation activity in yeast and could interact with the ubiquitin-conjugating enzymes E2_(s).An in vitro ubiquitination assay verified that TaGW2L possessed a similar E3 ligase activity to TaGW2.Overexpression of the TaGW2L-7A homoeolog in wheat led to a significantly earlier heading date under both natural conditions and long-day conditions.Transcriptome analysis revealed that multiple known genes positively regulating wheat heading were significantly upregulated in the TaGW2L-7A-overexpression plants compared with the wild-type control.Together,our findings shed light on the role of TaGW2L in wheat heading date and provide potential applications of TaGW2L for the adaptation improvement of crops.

The Role of E3 Ligases in Macrophage-mediated Inflammation

Macrophages,existed in almost all organs of the body,are responsible for detecting tissue injury,pathogens,playing a key role in host defense against a variety of invading pathogens triggering inflammatory responses.Emerging evidence suggests that macrophage-mediated immune responses are efficiently regulated by the ubiquitination modification,which is responsible for normal immune responses.However,numerous studies indicates that the aberrant activation or inhibition of macrophage-mediated immune responses occurs in inflammation,mainly caused by dysregulated ubiquitination modification due to E3 ubiquitin ligases mutations or abnormal expression.Notably,E3 ubiquitin ligases,responsible for recognizing the substrates,are key enzymes in the ubiquitin proteasome system(UPS)composed of ubiquitin(Ub),ubiquitin-activating E1 enzymes,ubiquitin-conjugating E2 enzymes,E3 ubiquitin ligases,26S proteasome,and deubiquitinating enzymes.Intriguingly,several E3 ubiquitin ligases are involved in the regulation of some common signal pathways in macrophage-mediated inflammation,including Toll-like receptors(TLRs),nucleotide-binding oligomerization domain(NOD)-like receptors(NLRs),RIG-I-like receptors(RLRs),C-type lectin receptors(CLRs)and the receptor for advanced glycation end products(RAGE).Herein,we summarized the physiological and pathological roles of E3 ligases in macrophage-mediated inflammation,as well as the inhibitors and agonists targeting E3 ligases in macrophage mediated inflammation,providing the new ideas for targeted therapies in macrophage-mediated inflammation caused aberrant function of E3 ligases.

Genetic investigation of the ubiquitin-protein ligase E3A gene as putative target in Angelman syndrome

BACKGROUND Angelman syndrome(AS)is caused by maternal chromosomal deletions,imprinting defects,paternal uniparental disomy involving chromosome 15 and the ubiquitin-protein ligase UBE3A gene mutations.However the genetic basis remains unclear for several patients.AIM To investigate the involvement of UBE3A gene in AS and identifying new potential genes using exome sequencing.METHODS We established a cohort study in 50 patients referred to Farhat Hached University Hospital between 2006 and 2021,with a strong suspicion of AS and absence of chromosomal aberrations.The UBE3A gene was screened for mutation detection.Two unrelated patients issued from consanguineous families were subjected to exome analysis.RESULTS We describe seven UBE3A variants among them 3 none previously described including intronic variants c.2220+14T>C(intron14),c.2507+43T>A(Exon15)and insertion in Exon7:c.30-47_30-46.The exome sequencing revealed 22 potential genes that could be involved in AS-like syndromes that should be investigated further.CONCLUSION Screening for UBE3A mutations in AS patients has been proven to be useful to confirm the diagnosis.Our exome findings could rise to new potential alternative target genes for genetic counseling.

Research Progress in Function and Regulation of E3 Ubiquitin Ligase SMURF1

Smad ubiquitylation regulatory factor 1(Smurf1)is an important homologous member of E6-AP C-terminus type E3 ubiquitin ligase.Initially,Smurf1 was reportedly involved in the negative regulation of the bone morphogenesis protein(BMP)pathway.After further research,several studies have confirmed that Smurf1 is widely involved in various biological processes,such as bone homeostasis regulation,cell migration,apoptosis,and planar cell polarity.At the same time,recent studies have provided a deeper understanding of the regulatory mechanisms of Smurf1’s expression,activity,and substrate selectivity.In our review,a brief summary of recent important biological functions and regulatory mechanisms of E3 ubiquitin ligase Smurf1 is proposed.

Plant E3 Ligases： Flexible Enzymes in a Sessile World

Since its discovery in the late 1970s, the ubiquitin proteasome pathway appears to be omnipresent in many research fields. Although originally discovered in animals, the pathway has a very central role in plants, which may be correlated to their sessile lifestyle. E3 ligases function as flexible and highly diverse key regulators within the path- way by targeting substrate proteins for ubiquitylation, and often proteolytic degradation via the 26S proteasome. This review provides a concise overview on the most common classes of E3 ligases so far described in plants, and emphasizes recent findings regarding these interesting and flexible enzymes and their diverse functions in plant biology.

Two VOZ transcription factors link an E3 ligase and an NLR immune receptor to modulate immunity in rice

Nucleotide-binding leucine-rich repeat(NLR)proteins play critical roles in plant immunity.However,how NLRs are regulated and activate defense signaling is not fully understood.The rice(Oryza sativa)NLR receptor Piz-t confers broad-spectrum resistance to the fungal pathogen Magnaporthe oryzae and the RING-type E3 ligase AVRPIZ-T INTERACTING PROTEIN 10(APIP10)negatively regulates Piz-t accumulation.In this study,we found that APIP10 interacts with two rice transcription factors,VASCULAR PLANT ONEZINC FINGER 1(OsVOZ1)and OsVOZ2,and promotes their degradation through the 26S proteasome pathway.OsVOZ1 displays transcriptional repression activity while OsVOZ2 confers transcriptional activation activity in planta.The osvoz1 and osvoz2 single mutants display modest but opposite M.oryzae resistance in the non-Piz-t background.However,the osvoz1 osvoz2 double mutant exhibits strong dwarfism and cell death,and silencing of both genes via RNA interference also leads to dwarfism,mild cell death,and enhanced resistance to M.oryzae in the non-Piz-t background.Both OsVOZ1 and OsVOZ2 interact with Piz-t.Double silencing of OsVOZ1 and OsVOZ2 in the Piz-t background decreases Piz-t protein accumulation and transcription,reactive oxygen species-dependent cell death,and resistance to M.oryzae containing AvrPiz-t.Taken together,these results indicate that OsVOZ1 and OsVOZ2 negatively regulate basal defense but contribute positively to Piz-t-mediated immunity.

Four Closely-related RING-type E3 Ligases,APD1-4,are Involved in Pollen Mitosis II Regulation in Arabidopsis

Ubiquitination of proteins is one of the critical regulatory mechanisms in eukaryotes. In higher plants, protein ubiquitination plays an essential role in many biological processes, including hormone signaling, photomorphogenesis, and pathogen defense. However, the roles of protein ubiquitination in the repro- ductive process are not clear. In this study, we identified four plant-specific RING-finger genes designated Aberrant_Pollen Development 1_ （APD1） to APD4, as regulators of pollen mitosis II （PMII） in Arabidopsis thaliana （L.）. The apdl apd2 double mutant showed a significantly increased percentage of bicellular-like pollen at the mature pollen stage. Further downregulation of the APD3 and APD4 transcripts in apdl apd2 by RNA interference （RNAi） resulted in more severe abnormal bicellular-like pollen phenotypes than in apdl apd2, suggesting that cell division was defective in male gametogenesis. All of the four genes were expressed in multiple stages at different levels during male gametophyte development. Confocal analysis using green florescence fusion proteins （GFP） GFP-APD1 and GFP-APD2 showed that APDs are associated with intracellular membranes. Furthermore, APD2 had E2-dependent E3 ligase activity in vitro, and five APD2-interacting proteins were identified. Our results suggest that these four genes may be involved, redundantly, in regulating the PMII process during male gametogenesis.

The RING E3 ligase CLG1 targets GS3 for degradation via the endosome pathway to determine grain size in rice

G-protein signaling and ubiquitin-dependent degradation are both involved in grain development in rice,but how these pathways are coordinated in regulating this process is unknown.Here,we show that Chang Li Geng 1(CLG1),which encodes an E3 ligase,regulates grain size by targeting the Gγprotein GS3,a negative regulator of grain length,for degradation.Overexpression of CLG1 led to increased grain length,while overexpression of mutated CLG1 with changes in three conserved amino acids decreased grain length.We found that CLG1 physically interacts with and ubiquitinats GS3which is subsequently degraded through the endosome degradation pathway,leading to increased grain size.Furthermore,we identified a critical SNP in the exon 3 of CLG1 that is significantly associated with grain size variation in a core collection of cultivated rice.This SNP results in an amino acid substitution from Arg to Ser at position 163 of CLG1 that enhances the E3 ligase activity of CLG1 and thus increases rice grain size.Both the expression level of CLG1 and the SNP CLG1163S may be useful variations for manipulating grain size in rice.

Identification of Arabidopsis MYB56 as a Nove Substrate for CRL3BPM E3 Ligases

Controlled stability of proteins is a highly efficient mechanism to direct diverse processes in living cells. A key regulatory system for protein stability is given by the ubiquitin proteasome pathway, which uses E3 ligases to mark specific proteins for degradation. In this work, MYB56 is identified as a novel target of a CULLIN3 （CUL3）-based E3 ligase. Its stability depends on the presence of MATH-BTB/POZ （BPM） proteins, which function as substrate adaptors to the E3 ligase. Genetic studies have indicated that MYB56 is a negative regulator of flowering, while BPMs positively affect this developmental program. The interaction between BPMs and MYB56 occurs at the promoter of FLOWERING LOCUS T （FT）, a key regulator in initiating flowering in Arabidopsis, and results in instability of MYB56. Overall the work establishes MYB transcription factors as substrates of BPM proteins, and provides novel information on components that participate in controlling flowering time in plants.

XBAT35, a Novel Arabidopsis RING E3 Ligase Exhibiting Dual Targeting of Its Splice Isoforms, Is Involved in Ethylene-Mediated Regulation of Apical Hook Curvature

The Arabidopsis XBAT35 is one of five structurally related ankyrin repeat-containing Really Interesting New Gene （RING） E3 ligases involved in ubiquitin-mediated protein degradation, which plays key roles in a wide range of cel- lular processes. Here, we show that the XBAT35 gene undergoes alternative splicing, generating two transcripts that are constitutively expressed in all plant tissues. The two splice variants derive from an exon skipping event that excludes an in-frame segment from the XBAT35 precursor mRNA, giving rise to two protein isoforms that differ solely in the presence of a nuclear localization signal （NLS）. Transient expression assays indicate that the isoform lacking the NLS localizes in the cytoplasm of plant cells, whereas the other is targeted to the nucleus, accumulating in nuclear speckles. Both isoforms are functional E3 ligases, as assessed by in vitro ubiquitination assays. Two insertion mutant alleles and RNA-interference （RNAi） silencing lines for XBAT35 display no evident phenotypes under normal growth conditions, but exhibit hyper- sensitivity to the ethylene precursor 1-aminocyclopropane-l-carboxylate （ACC） during apical hook exaggeration in the dark, which is rescued by an inhibitor of ethylene perception. Independent expression of each XBAT35 splice variant in the mutant background indicates that the two isoforms may differentially contribute to apical hook formation but are both functional in this ethylene-mediated response. Thus, XBAT35 defines a novel player in ethylene signaling involved in negatively regulating apical hook curvature, with alternative splicing controlling dual targeting of this E3 ubiquitin ligase to the nuclear and cytoplasmic compartments.

Functional analyses of an E3 ligase gene AIP2 from wheat in Arabidopsis revealed its roles in seed germination and pre‐harvest sprouting

Pre‐harvest sprouting（PHS） seriously affects wheat yield and quality of the grain. ABI3 is a key factor in the activation of seed development and repression of germination in Arabidopsis. An ABI3‐interacting protein（AIP2） could polyubiquitinate ABI3, impair seed dormancy and promote seed germination in Arabidopsis. In this study,two wheat AIP2 genes, TaAIP2A and TaAIP2B, were isolated.Subcellular localization assay and yeast two‐hybrid analysis revealed that TaAIP2A and TaAIP2B may function through interaction with wheat Viviporous‐1（TaVp1）. The transcripts TaAIP2A and TaAIP2B were more abundant in wheat PHS susceptible cultivars than that of resistant ones, and decreased gradually following seed development. Expression of TaAIP2A and TaAIP2B in Arabidopsis aip2‐1 mutant lines resulted in earlier flowering, promotion of seed germination,and reduced ABA sensitivity, respectively, somehow mimicking the phenotype of the wild type, with TaAIP2B having a stronger role in these aspects. Furthermore, the expression ofupstream genes ABI1 and ABI2 were upregulated, whereas that of downstream genes ABI3 and ABI5 were downregulated in both TaAIP2A and TaAIP2B complemented lines upon ABA treatment. These results suggested that wheat AIP2s could negatively regulate the ABA signaling pathway and play important roles in seed germination, and thus wheat PHS resistance finally.

The E3 ligase XBAT35 mediates thermoresponsive hypocotyl growth by targeting ELF3 for degradation in Arabidopsis

Plants are capable of coordination of their growth and development with ambient temperatures.EARLY FLOWERING3(ELF3), an essential component of the plant circadian clock, is also involved in ambient temperature sensing, as well as in inhibiting the expression and protein activity of the thermoresponsive regulator phytochrome interacting factor4(PIF4). The ELF3 activity is subjected to attenuation in response to warm temperature;however,how the protein level of ELF3 is regulated at warm temperature remains less understood. Here, we report that the E3 ligase XB3 ORTHOLOG 5 IN ARABIDOPSIS THALIANA, XBAT35, mediates ELF3 degradation. XBAT35 interacts with ELF3 and ubiquitinates ELF3. Loss-of-function mutation of XBAT35 increases the protein level of ELF3 and confers a short-hypocotyl phenotype under warm temperature conditions. Thus, our findings establish that XBAT35 mediates ELF3 degradation to lift the inhibition of ELF3 on PIF4 for promoting thermoresponsive hypocotyl growth in plants.

MYB106 is a negative regulator and a substrate for CRL3^(BPM) E3 ligase in regulating flowering time in Arabidopsis thaliana

Flowering time is crucial for successful reproduction in plants, the onset and progression of which are strictly controlled. However, flowering time is a complex and environmentally responsive history trait and the underlying mechanisms still need to be fully characterized. Post-translational regulation of the activities of transcription factors(TFs) is a dynamic and essential mechanism for plant growth and development. CRL3 BPME3 ligase is a CULLIN3-based E3 ligase involved in orchestrating protein stability via the ubiquitin proteasome pathway. Our study shows that the mutation of MYB106 induced early flowering phenotype while over-expression of MYB106 delayed Arabidopsis flowering. Transcriptome analysis of myb106 mutants reveals 257 differentially expressed genes between wild type and myb106-1 mutants, including Flowering Locus T(FT) which is related to flowering time. Moreover, in vitro electrophoretic mobility shift assays(EMSA), in vivo chromatin immunoprecipitation quantitative polymerase chain reaction(ChIP-q PCR) assays and dual luciferase assays demonstrate that MYB106 directly binds to the promoter of FT to suppress its expression. Furthermore, we confirm that MYB106 interacts with BPM proteins which are further identified by CRL3 BPME3 ligases as the substrate. Taken together, we have identified MYB106 as a negative regulator in the control of flowering time and a new substrate for CRL3 BPM E3 ligases in Arabidopsis.

An engineered platform for reconstituting functional multisubunit SCF E3 ligase in vitro

Multisubunit SKP1/Cullin1/F-box(SCF)E3 ligases play essential roles in regulating the stability of crucial regulatory factors and controlling growth and development in eukaryotes.Detecting E3 ligase activity in vitro is important forexploring the molecular mechanism of protein ubiquitination.However,in vitro ubiquitination assay systems for multisubunit E3 ligases remain difficult to achieve,especially in plants,mainly owing to difficulties in achieving active components of multisubunit E3 ligases with high purity and characterizing specific E2 and E3 pairs.In this study,we characterized components of the rice ScFDiwARF3(SCFDs)E3 ligase,screened the coordinated E2,and reconstituted active ScFD3 E3 ligase in vitro.We further engineered SCFD3 E3 ligase using a fused SKP1-Cullin1-RBX1(eSCR)protein and found that both the wild-type SCFD3 E3 ligase and the engineered SCFD3 E3 ligase catalyzed ubiquitination of the substrate D53,which is the key transcriptional repressor in strigolactone signaling.Finally,we replaced D3 with other F-box proteins from rice and humans and reconstituted active escF E3 ligases,including escFaID2,escFBxL1s,and escFcDC4 E3 ligases.Our work reconstitutes functional SCF E3 ligases in vitro and generates an engineered system with interchangeable F-box proteins,providing a powerful platform for studying the mechanisms of multisubunit SCF E3 ligases in eukaryotes.

The Ubiquitin E3 Ligase PUB17 Positively Regulates Immunity by Targeting a Negative Regulator, KH17, for Degradation

Ubiquitination is a post-translational modification that regulates many processes in plants.Several ubiquitin E3 ligases act as either positive or negative regulators of immunity by promoting the degradation of different substrates.StPUB17 is an E3 ligase that has previously been shown to positively regulate immunity to bacteria,fungi and oomycetes,including the late blight pathogen Phytophthora infestans.Silencing of StPUB17 promotes pathogen colonization and attenuates Cf4/avr4 cell death.Using yeast-2-hybrid and co-immunoprecipitation we identified the putative K-homology(KH)RNA-binding protein(RBP),StKH17,as a candidate substrate for degradation by StPUB17.StKH17 acts as a negative regulator of immunity that promotes P.infestans infection and suppresses specific immune pathways.A KH RBP domain mutant of StKH17(StKH17GDDG)is no longer able to negatively regulate immunity,indicating that RNA binding is likely required for StKH17 function.As StPUB17 is a known target of the ubiquitin E3 ligase,StPOB1,we reveal an additional step in an E3 ligase regulatory cascade that controls plant defense.

Drosophila ubiquitin E3 ligase dSmurf is required for synapse remodeling and axon pruning by glia

Animal behaviors and higher-order functions rely on complex neural circuits built by synaptic connections （synapses） to deliver messages among different brain cells. As the major mediator in the nervous system, neurons communicate via synapses, which undergo constant structural remodeling with strict regulation.

Human RING finger protein ZNF645 is a novel testis-specific E3 ubiquitin ligase

A large number of testis-specific genes are involved in the complex process of mammalian spermatogenesis. Identification of these genes and their roles is important for understanding the mechanisms underlying spermatogenesis. Here we report on a novel human RING finger protein, ZNF645, which contains a C3HC4 RING finger domain, a C2H2 zinc-finger domain, and a proline-rich region, indicating that it has a structure similar to that of the c-Cbl-like protein Hakai. ZNF645 was exclusively expressed in normal human testicular tissue. Immunohistochemical analysis confirmed that ZNF645 protein was present in spermatocytes, round and elongated spermatids, and Leydig cells. Immunofluorescence staining of mature sperms further showed that the ZNF645 protein was localized over the postacrosomal perinuclear theca region and the entire length of sperm tail. An in vitro ubiquitination assay indicated that the RING finger domain of the ZNF645 protein had E3 ubiquitin ligase activity. Therefore, we suggest that ZNF645 might act as an E3 ubiquitin-protein ligase and play a role in human sperm production and quality control.

Immune regulation by protein ubiquitination: roles of the E3 ligases VHL and Itch

Protein ubiquitination is an important means of posttranslational modification which plays an essential role in the regulation of various aspects of leukocyte development and function. The specificity of ubiquitin tagging to a protein substrate is determined by E3 ubiquitin ligases via defined E3-substrate interactions. In this review, we will focus on two E3 ligases, VHL and Itch, to discuss the latest progress in understanding their roles in the differentiation and function of CD4+ T helper cell subsets, the stability of regulatory T cells, effector function of CD8+ T cells, as well as the development and maturation of innate lymphoid cells. The biological implications of these E3 ubiquitin ligases will be highlighted in the context of normal and dysregulated immune responses including the control of homeostasis, inflammation, auto-immune responses and anti-tumor immunity. Further elucidation of the ubiquitin system in immune cells will help in the design of new therapeutic interventions for human immunological diseases and cancer.

Functional Characterization of the Apple RING E3 Ligase MdMIEL1 in Transgenic Arabidopsis

E3 ubiquitin ligases are involved in various physiological processes,and they play pivotal roles in growth and development.In this study,we identified a previously unknown gene in the apple fruit(Malus×domestica)and named it MdMIEL1.The MdMIEL1 gene encoded a protein that contained a zinc-finger domain at its N-terminus and a RING-finger motif at its C-terminus.To investigate MdMIEL1 functions,we generated transgenic Arabidopsis lines expressing the MdMIEL1 gene under the control of the Cauliflower mosaic virus 35S promoter.Interestingly,ectopic expression of MdMIEL1 in Arabidopsis produced multiple phenotypes,including early germination,early flowering and a lateral root number increase relative to wild-type plants.Further analysis indicated that MdMIEL1 regulated lateral root initiation by increasing auxin accumulation in the roots.In a word,these results suggest that,MdMIEL1 as a novel RING-finger ubiquitin ligase influences plant growth and development,and highlight that MdMIEL1 regulates lateral root growth.