RING-type E3 Ligase Research Articles

Determinants of chemoselectivity in ubiquitination by the J2 family of ubiquitin-conjugating enzymes.

Ubiquitin-conjugating enzymes (E2) play a crucial role in the attachment of ubiquitin to proteins. Together with ubiquitin ligases (E3), they catalyze the transfer of ubiquitin (Ub) onto lysines with high chemoselectivity. A subfamily of E2s, including yeast Ubc6 and human Ube2J2, also mediates noncanonical modification of serines, but the structural determinants for this chemical versatility remain unknown. Using a combination of X-ray crystallography, molecular dynamics (MD) simulations, and reconstitution approaches, we have uncovered a two-layered mechanism that underlies this unique reactivity. A rearrangement of the Ubc6/Ube2J2 active site enhances the reactivity of the E2-Ub thioester, facilitating attack by weaker nucleophiles. Moreover, a conserved histidine in Ubc6/Ube2J2 activates a substrate serine by general base catalysis. Binding of RING-type E3 ligases further increases the serine selectivity inherent to Ubc6/Ube2J2, via an allosteric mechanism that requires specific positioning of the ubiquitin tail at the E2 active site. Our results elucidate how subtle structural modifications to the highly conserved E2 fold yield distinct enzymatic activity.

The Arabidopsis E3 ubiquitin ligase DOA10A promotes localization of abscisic acid (ABA) receptors to the membrane through mono-ubiquitination in ABA signaling.

The endoplasmic reticulum-associated degradation (ERAD) system eliminates misfolded and short-lived proteins to maintain physiological homeostasis in the cell. We have previously reported that ERAD is involved in salt tolerance in Arabidopsis. Given the central role of the phytohormone abscisic acid (ABA) in plant stress responses, we sought to identify potential intersections between the ABA and the ERAD pathways in plant stress response. By screening for the ABA response of a wide array of ERAD mutants, we isolated a gain-of-function mutant, doa10a-1, which conferred ABA hypersensitivity to seedlings. Genetic and biochemical assays showed that DOA10A is a functional E3 ubiquitin ligase which, by acting in concert with specific E2 enzymes, mediates mono-ubiquitination of the ABA receptor, followed by their relocalization to the plasma membrane. This in turn leads to enhanced ABA perception. In summary, we report here the identification of a novel RING-type E3 ligase, DOA10A, which regulates ABA perception by affecting the localization and the activity of ABA receptors through their mono-ubiquitination.

Interaction of the cyclin-dependent kinase inhibitor GhKRP6 with RING-Type E3 ligase influences the fiber length in Upland cotton

Upland cotton (Gossypium hirsutum L.) is the major crop producing renewable fiber for textiles. Cotton fiber length is an economically important trait that affects the quality of the yarn. The Upland cotton multi-parent advanced generation intercross (MAGIC) population was developed for genetics research to understand how fiber traits can be improved without reducing fiber yield. A genome-wide association study of the MAGIC population identified a significant fiber length QTL on Chromosome D11 (qFL-D11–1). To understand how the qFL-D11–1 regulates fiber length, we developed F5 lines by crossing the longest and the shortest fiber recombinant inbred lines (RILs) of the MAGIC population to establish lines with parental haplotypes at the qFL-D11–1 but segregating for other loci. The F5 lines and parental RILs were sequenced to detect exchanges from parents' genomic regions in the progeny. One segregating genomic region in the F5 lines coincided with qFL-D12–1, which additively affected fiber length. Correlation analysis of the expression patterns of genes from the two QTLs with the fiber lengths in the MAGIC population showed the highest correlation of the RING-type ubiquitin E3 ligase (GhUbE3) from qFL-D12–1. Kip-related protein-6 (KRP6) is the candidate gene from the qFL-D11–1. A yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated a physical interaction between GhKRP6 (Ghir_D11G020340) and GhUbE3 (Ghir_D12G006080). RNAseq showed that the GhUbE3 expression is reciprocal to the interacting GhKRP6 during fiber development. Virus-induced gene silencing (VIGS) of GhKRP6 increased fiber length. The data indicates that GhUbE3 controls the dose of GhKRP6's inhibitory activity and, therefore, the duration of fiber elongation.

Mechanism of nucleosomal H2A K13/15 monoubiquitination and adjacent dual monoubiquitination by RNF168.

The DNA damage repair regulatory protein RNF168, a monomeric RING-type E3 ligase, has a crucial role in regulating cell fate and DNA repair by specific and efficient ubiquitination of the adjacent K13 and K15 (K13/15) sites at the H2A N-terminal tail. However, understanding how RNF168 coordinates with its cognate E2 enzyme UbcH5c to site-specifically ubiquitinate H2A K13/15 has long been hampered by the lack of high-resolution structures of RNF168 and UbcH5c~Ub (ubiquitin) in complex with nucleosomes. Here we developed chemical strategies and determined the cryo-electron microscopy structures of the RNF168-UbcH5c~Ub-nucleosome complex captured in transient H2A K13/15 monoubiquitination and adjacent dual monoubiquitination reactions, providing a 'helix-anchoring' mode for monomeric E3 ligase RNF168 on nucleosome in contrast to the 'compass-binding' mode of dimeric E3 ligases. Our work not only provides structural snapshots of H2A K13/15 site-specific monoubiquitination and adjacent dual monoubiquitination but also offers a near-atomic-resolution structural framework for understanding pathogenic amino acid substitutions and physiological modifications of RNF168.

TRAF7 determines circadian period through ubiquitination and degradation of DBP.

D-site binding protein, DBP, is a clock-controlled transcription factor and drives daily rhythms of physiological processes through the regulation of an array of genes harboring a DNA binding motif, D-box. DBP protein levels show a circadian oscillation with an extremely robust peak/trough ratio, but it is elusive how the temporal pattern is regulated by post-translational regulation. In this study, we show that DBP protein levels are down-regulated by the ubiquitin-proteasome pathway. Analysis using 19 dominant-negative forms of E2 enzymes have revealed that UBE2G1 and UBE2T mediate the degradation of DBP. A proteomic analysis of DBP-interacting proteins and database screening have identified Tumor necrosis factor Receptor-Associated Factor 7 (TRAF7), a RING-type E3 ligase, that forms a complex with UBE2G1 and/or UBE2T. Ubiquitination analysis have revealed that TRAF7 enhances K48-linked polyubiquitination of DBP in cultured cells. Overexpression of TRAF7 down-regulates DBP protein level, while knockdown of TRAF7 up-regulates DBP in cultured cells. Knockout of TRAF7 in NIH3T3 cells have revealed that TRAF7 mediates the time-of-the-day-dependent regulation of DBP levels. Furthermore, TRAF7 has a period-shortening effect on the cellular clock. Together, TRAF7 plays an important role in circadian clock oscillation through destabilization of DBP.

The RING-Type E3 Ligase BOI Interacts with EXO70E2 and Mediates Its Ubiquitination in Arabidopsis.

The exocyst is a hetero-octameric complex that exhibits significant functional diversity in regulating biological processes and defense responses. In plants, the EXO70 proteins are important components of the exocyst complex and are involved in membrane trafficking, biotic and abiotic interactions, as well as cell wall formation. A previous study has indicated that a member of the EXO subfamily, EXO70E2, interacts with RIN4 to mediate plant immunity. In this study, we found that EXO70E2 interacts with the RING-type E3 ligase Botrytis susceptible1 interactor (BOI), and the C-terminal domain of BOI is necessary for its interaction with EXO70E2. Moreover, the protein level of EXO70E2 was degraded and ubiquitinated by BOI in vitro. Collectively, our study reveals a mechanism for regulating the stability of EXO70E2 by a RING-type E3 ligase BOI-mediated ubiquitination.

Pepper RING-Type E3 Ligase CaFIRF1 Negatively Regulates the Protein Stability of Pepper Stress-Associated Protein, CaSAP14, in the Dehydration Stress Response.

As part of the cellular stress response in plants, the ubiquitin-proteasome system (UPS) plays a crucial role in regulating the protein stability of stress-related transcription factors. Previous study has indicated that CaSAP14 is functionally involved in enhancing pepper plant tolerance to dehydration stress by modulating the expression of downstream genes. However, the comprehensive regulatory mechanism underlying CaSAP14 remains incompletely understood. Here, we identified a RING-type E3 ligase, CaFIRF1, which interacts with and ubiquitinates CaSAP14. Pepper plants with silenced CaFIRF1 exhibited a dehydration-tolerant phenotype when subjected to dehydration stress, while overexpression of CaFIRF1 in pepper and Arabidopsis resulted in reduced dehydration tolerance. Co-silencing of CaFIRF1 and CaSAP14 in pepper increased sensitivity to dehydration, suggesting that CaFIRF1 acts upstream of CaSAP14. A cell-free degradation analysis demonstrated that silencing of CaFIRF1 led to decreased CaSAP14 protein degradation, implicating CaFIRF1 in the regulation of CaSAP14 protein via the 26S proteasomal degradation pathway. Our findings suggest a mechanism by which CaFIRF1 mediates the ubiquitin-dependent proteasomal degradation of CaSAP14, thereby influencing the response of pepper plants to dehydration stress.

The RING-type E3 ligase, TaFRFP, regulates flowering by controlling a salicylic acid-mediated floral promotion

The initiation of transition to flowering is carefully managed by endogenous and environmental cues, which is critical for flowering plant reproductive success. Here, we found that wheat RING-type E3 ligase TaFRFP was highly expressed from the double ridge to degeneration stage (WS2.5-WS9). TaFRFP is localized in the nucleus and has E3 ligase activity in vitro. TaFRFP overexpression in Arabidopsis resulted in an early flowering phenotype, but to a lesser extent, under short-day conditions. Under the SA-treated condition, overexpression of TaFRFP shows higher root growth and has more accumulation of SA contents. A proteomic comparison revealed that the amount of FRL4A protein, a FRIGIDA LIKE 4 A, was considerably lower in SA-treated TaFRFP seedlings compared to normal condition. We further found that TaFRFP directly interacts with FRL4A in the nucleus and recruits it to the FLC locus in Arabidopsis. Moreover, an ubiquitination assay showed that TaFRPF physically interact and ubiquitinates TaFRL as a substrate. Our findings support the concept that the TaFRFP E3 ligase works as a positive regulator, and that the ubiquitination of its substrate proteins plays a significant role in controlling flowering time via an SA-dependent pathway.

Pepper SUMO protease CaDeSI2 positively modulates the drought responses via deSUMOylation of clade A PP2C CaAITP1.

Posttranslational modification of multiple ABA signaling components is an essential process for the adaptation and survival of plants under stress conditions. In our previous study, we established that the pepper group A PP2C protein CaAITP1, one of the core components of ABA signaling, undergoes ubiquitination mediated by the RING-type E3 ligase CaAIRE1. In this study, we discovered an additional form of regulation mediated via the SUMOylation of CaAITP1. Pepper plants subjected to drought stress were characterized by reductions in both the stability and SUMOylation of CaAITP1 protein. Moreover, we identified a SUMO protease, Capsicum annuum DeSUMOylating Isopeptidase 2 (CaDeSI2), as a new interacting partner of CaAITP1. In vitro and in vivo analyses revealed that CaAITP1 is deSUMOylated by CaDeSI2. Silencing of CaDeSI2 in pepper plants led to drought-hypersensitive and ABA-hyposensitive phenotypes, whereas overexpression of CaDeSI2 in transgenic Arabidopsis plants resulted in the opposite phenotypes. Importantly, we found that the CaAITP1 protein was stabilized in response to the silencing of CaDeSI2, and CaDeSI2 and CaAITP1 co-silenced pepper plants were characterized by drought-tolerant phenotypes similar to those observed in CaAITP1-silenced pepper. Collectively, our findings indicate that CaDeSI2 reduces the stability of CaAITP1 via deSUMOylation, thereby positively regulating drought tolerance.

Rab7 GTPase-Mediated stress signaling enhances salinity tolerance in AlRabring7 tobacco transgenics by modulating physio-biochemical parameters

The RING-type E3 ligases play a significant role in stress signaling, primarily through post-translational regulation. Ubiquitination is a crucial post-translational modification that regulates the turnover and activity of proteins. The overexpression of AlRabring7, RING-HC E3 Ub ligase in tobacco provides insights into the regulation of salinity and ABA signaling in transgenic tobacco. The seed germination potential of AlRabring7 transgenics was higher than WT, with NaCl and ABA treatments. The transgenics showed improved morpho-physio-biochemical parameters in response to salinity and ABA treatments. The photosynthetic pigments, soluble sugars, reducing sugars and proline increased in transgenics in response to NaCl and ABA treatments. The decreased ROS accumulation in transgenics on NaCl and ABA treatments can be co-related to improved activity of enzymatic and non-enzymatic antioxidants. The potential of transgenics to maintain ABA levels with ABA treatment, highlights the active participation of ABA feedback loop mechanism. Interestingly, the ability of AlRabring7 transgenics to upregulate Rab7 protein, suggests its role in facilitating vacuolar transport. Furthermore, the improved potassium accumulation and reduced sodium content indicate an efficient ion regulation mechanism in transgenic plants facilitating higher stomatal opening. The expression of downstream ion transporter (NbNHX1 and NbVHA1), ABA signaling (NbABI2 and NbABI5) and vesicle trafficking (NbMON1) responsive genes were upregulated with stress. The present study, reports that AlRabring7 participates in maintaining vacuolar transport, ion balance, ROS homeostasis, stomatal regulation through activation of Rab7 protein and regulation of downstream stress-responsive during stress. This emphasizes the potential of AlRabring7 gene for improved performance and resilience in challenging environments.

Population-level exploration of alternative splicing and its unique role in controlling agronomic traits of rice.

Alternative splicing (AS) plays crucial roles in regulating various biological processes in plants. However, the genetic mechanisms underlying AS and its role in controlling important agronomic traits in rice (Oryza sativa) remain poorly understood. In this study, we explored AS in rice leaves and panicles using the rice minicore collection. Our analysis revealed a high level of transcript isoform diversity, with approximately one-fifth of the potential isoforms acting as major transcripts in both tissues. Regarding the genetic mechanism of AS, we found that the splicing of 833 genes in the leaf and 1,230 genes in the panicle was affected by cis-genetic variation. Twenty-one percent of these AS events could only be explained by large structural variations. Approximately 77.5% of genes with significant splicing quantitative trait loci (sGenes) exhibited tissue-specific regulation, and AS can cause 26.9% (leaf) and 23.6% (panicle) of sGenes to have altered, lost, or gained functional domains. Additionally, through splicing-phenotype association analysis, we identified phosphate-starvation-induced RING-type E3 ligase (OsPIE1; LOC_Os01g72480), whose splicing ratio was significantly associated with plant height. In summary, this study provides an understanding of AS in rice and its contribution to the regulation of important agronomic traits.

Integration of C3H15-mediated transcriptional and post-transcriptional regulation confers plant thermotolerance in Arabidopsis.

Heat stress is an environmental factor that significantly threatens crop production worldwide. Nevertheless, the molecular mechanisms governing plant responses to heat stress are not fully understood. Plant zinc finger CCCH proteins have roles in stress responses as well as growth and development through protein-RNA, protein-DNA, and protein-protein interactions. Here, we reveal an integrated multi-level regulation of plant thermotolerance that is mediated by the CCCH protein C3H15 in Arabidopsis. Heat stress rapidly suppressed C3H15 transcription, which attenuated C3H15-inhibited expression of its target gene HEAT SHOCK TRANSCRIPTION FACTOR A2 (HSFA2), a central regulator of heat stress response (HSR), thereby activating HEAT SHOCK COGNATE 70 (HSC70.3) expression. The RING-type E3 ligase MED25-BINDING RING-H2 PROTEIN 2 (MBR2) was identified as an interacting partner of C3H15. The mbr2 mutant was susceptible to heat stress compared to wild-type plants, whereas plants overexpressing MBR2 showed increased heat tolerance. MBR2-dependent ubiquitination mediated the degradation of phosphorylated C3H15 protein in the cytoplasm, which was enhanced by heat stress. Consistently, heat sensitivities of C3H15 overexpression lines increased in MBR2 loss-of-function and decreased in MBR2 overexpression backgrounds. Heat stress-induced accumulation of HSC70.3 promoted MBR2-mediated degradation of C3H15 protein, implying that an auto-regulatory loop involving C3H15, HSFA2, and HSC70.3 regulates HSR. Heat stress also led to the accumulation of C3H15 in stress granules (SGs), a kind of cytoplasmic RNA granule. This study advances our understanding of the mechanisms plants use to respond to heat stress, which will facilitate technologies to improve thermotolerance in crops.

Dynamic modulation of nodulation factor receptor levels by phosphorylation-mediated functional switch of a RING-type E3 ligase during legume nodulation

The precise control of receptor levels is crucial for initiating cellular signaling transduction in response to specific ligands; however, such mechanisms regulating nodulation factor (NF) receptor (NFR)-mediated perception of NFs to establish symbiosis remain unclear. In this study, we unveil the pivotal role of the NFR-interacting RING-type E3 ligase 1 (NIRE1) in regulating NFR1/NFR5 homeostasis to optimize rhizobial infection and nodule development in Lotus japonicus. We demonstrated that NIRE1 has a dual function in this regulatory process. It associates with both NFR1 and NFR5, facilitating their degradation through K48-linked polyubiquitination before rhizobial inoculation. However, following rhizobial inoculation, NFR1 phosphorylates NIRE1 at a conserved residue, Tyr-109, inducing a functional switch in NIRE1, which enables NIRE1 to mediate K63-linked polyubiquitination, thereby stabilizing NFR1/NFR5 in infected root cells. The introduction of phospho-dead NIRE1Y109F leads to delayed nodule development, underscoring the significance of phosphorylation at Tyr-109 in orchestrating symbiotic processes. Conversely, expression of the phospho-mimic NIRE1Y109E results in the formation of spontaneous nodules in L. japonicus, further emphasizing the critical role of the phosphorylation-dependent functional switch in NIRE1. In summary, these findings uncover a fine-tuned symbiotic mechanism that a single E3 ligase could undergo a phosphorylation-dependent functional switch to dynamically and precisely regulate NF receptor protein levels.

DGS1 improves rice disease resistance by elevating pathogen-associated molecular pattern-triggered immunity.

The online version contains supplementary material available at 10.1007/s42994-024-00137-9.

E3 Ubiquitin Ligase Uhrf2 Knockout Reveals a Critical Role in Social Behavior and Synaptic Plasticity in the Hippocampus.

The hippocampal formation, particularly the CA2 subregion, is critical for social memory formation and memory processing, relying on synaptic plasticity-a fundamental mechanism by which synapses strengthen. Given the role of the ubiquitin-proteasome system (UPS) in various nervous system processes, including learning and memory, we were particularly interested in exploring the involvement of RING-type ubiquitin E3 ligases, such as UHRF2 (NIRF), in social behavior and synaptic plasticity. Our results revealed altered social behavior in mice with systemic Uhrf2 knockout, including changes in nest building, tube dominance, and the three-chamber social novelty test. In Uhrf2 knockout mice, the entorhinal cortex-CA2 circuit showed significant reductions in synaptic plasticity during paired-pulse facilitation and long-term potentiation, while the inability to evoke synaptic plasticity in the Schaffer-collateral CA2 synapses remained unaffected. These changes in synaptic plasticity correlated with significant changes in gene expression including genes related to vesicle trafficking and transcriptional regulation. The effects of Uhrf2 knockout on synaptic plasticity and the observed gene expression changes highlight UHRF2 as a regulator of learning and memory processes at both the cellular and systemic levels. Targeting E3 ubiquitin ligases, such as UHRF2, may hold therapeutic potential for memory-related disorders, warranting further investigation.

A pepper RING-finger E3 ligase, CaFIRF1, negatively regulates the high-salt stress response by modulating the stability of CaFAF1.

Controlling protein stability or degradation via the ubiquitin-26S proteasome system is a crucial mechanism in plant cellular responses to stress conditions. Previous studies have revealed that the pepper FANTASTIC FOUR-like gene, CaFAF1, plays a positive role in salt tolerance and that, in this process, CaFAF1 protein degradation is delayed. Here, we sought to isolate the E3 ligases potentially responsible for modulating CaFAF1 protein stability in response to salt stress. The pepper RING-type E3 ligase CaFIRF1 (Capsicum annuum FAF1 Interacting RING Finger protein 1) was found to interact with and ubiquitinate CaFAF1, leading to the degradation of CaFAF1 proteins. In response to high-salt treatments, CaFIRF1-silenced pepper plants exhibited tolerant phenotypes. In contrast, co-silencing of CaFAF1 and CaFIRF1 led to increased sensitivity to high-salt treatments, revealing that CaFIRF1 functions upstream of CaFAF1. A cell-free degradation analysis showed that high-salt treatment suppressed CaFAF1 protein degradation via the 26S proteasome pathway, in which CaFIRF1 is functionally involved. In addition, an in vivo ubiquitination assay revealed that CaFIRF1-mediated ubiquitination of CaFAF1 proteins was reduced by high-salt treatment. Taken together, these findings suggest that the degradation of CaFAF1 mediated by CaFIRF1 has a critical role in pepper plant responses to high salinity.

Arabidopsis HECT and RING-type E3 Ligases Promote MAPKKK18 Degradation to Regulate Abscisic Acid Signaling.

Mitogen-activated protein kinase (MAPK) cascades are conserved signaling pathways that transduce extracellular signals into diverse cellular responses. Arabidopsis MAPKKK18 is a component of the MAPKKK17/18-MKK3-MPK1/2/7/14 cascades, which play critical roles in abscisic acid (ABA) signaling, drought tolerance and senescence. A very important aspect of MAP kinase signaling is both its activation and its termination, which must be tightly controlled to achieve appropriate biological responses. Recently, the ubiquitin-proteasome system (UPS) has received increasing attention as a key mechanism for maintaining the homeostasis of MAPK cascade components and other ABA signaling effectors. Previous studies have shown that the stability of MAPKKK18 is regulated by the UPS via the ABA core pathway. Here, using multiple proteomic approaches, we found that MAPKKK17/18 turnover is tightly controlled by three E3 ligases, UPL1, UPL4 and KEG. We also identified lysines 154 and 237 as critical for MAPKKK18 stability. Taken together, this study sheds new light on the mechanism that controls MAPKKK17/18 activity and function.

BioE3 identifies specific substrates of ubiquitin E3 ligases

Hundreds of E3 ligases play a critical role in recognizing specific substrates for modification by ubiquitin (Ub). Separating genuine targets of E3s from E3-interactors remains a challenge. We present BioE3, a powerful approach for matching substrates to Ub E3 ligases of interest. Using BirA-E3 ligase fusions and bioUb, site-specific biotinylation of Ub-modified substrates of particular E3s facilitates proteomic identification. We show that BioE3 identifies both known and new targets of two RING-type E3 ligases: RNF4 (DNA damage response, PML bodies), and MIB1 (endocytosis, autophagy, centrosome dynamics). Versatile BioE3 identifies targets of an organelle-specific E3 (MARCH5) and a relatively uncharacterized E3 (RNF214). Furthermore, BioE3 works with NEDD4, a HECT-type E3, identifying new targets linked to vesicular trafficking. BioE3 detects altered specificity in response to chemicals, opening avenues for targeted protein degradation, and may be applicable for other Ub-likes (UbLs, e.g., SUMO) and E3 types. BioE3 applications shed light on cellular regulation by the complex UbL network.

RING-type E3 ligase BnaJUL1 ubiquitinates and degrades BnaTBCC1 to regulate drought tolerance in Brassica napus L.

Drought stress poses a persistent threat to field crops and significantly limits global agricultural productivity. Plants employ ubiquitin-dependent degradation as a crucial post-translational regulatory mechanism to swiftly adapt to changing environmental conditions. JUL1 is a RING-type E3 ligase related to drought stress in Arabidopsis. In this study, we explored the function of BnaJUL1 (a homologous gene of JUL1 in Brassica napus) and discovered a novel gene BnaTBCC1 participating in drought tolerance. First, we utilised BnaJUL1-cri materials through the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 system. Second, we confirmed that BnaJUL1 regulated drought tolerance through the drought tolerance assay and transcriptome analysis. Then, we identified a series of proteins interacting with BnaJUL1 through yeast library screening, including BnaTBCC1 (a tubulin binding cofactor C domain-containing protein); whose homologous gene TBCC1 knockdown mutants (tbcc1-1) exhibited ABA-sensitive germination in Arabidopsis, we then confirmed the involvement of BnaTBCC1 in drought tolerance in both Arabidopsis and Brassica. Finally, we established that BnaJUL1 could ubiquitinate and degrade BnaTBCC1 to regulate drought tolerance. Consequently, our study unveils BnaJUL1 as a novel regulator that ubiquitinates and degrades BnaTBCC1 to modulate drought tolerance and provided desirable germplasm for further breeding of drought tolerance in rapeseed.

Molecular basis for PHF7-mediated ubiquitination of histone H3.

The RING-type E3 ligase has been known for over two decades, yet its diverse modes of action are still the subject of active research. Plant homeodomain (PHD) finger protein 7 (PHF7) is a RING-type E3 ubiquitin ligase responsible for histone ubiquitination. PHF7 comprises three zinc finger domains: an extended PHD (ePHD), a RING domain, and a PHD. While the function of the RING domain is largely understood, the roles of the other two domains in E3 ligase activity remain elusive. Here, we present the crystal structure of PHF7 in complex with the E2 ubiquitin-conjugating enzyme (E2). Our structure shows that E2 is effectively captured between the RING domain and the C-terminal PHD, facilitating E2 recruitment through direct contact. In addition, through in vitro binding and functional assays, we demonstrate that the N-terminal ePHD recognizes the nucleosome via DNA binding, whereas the C-terminal PHD is involved in histone H3 recognition. Our results provide a molecular basis for the E3 ligase activity of PHF7 and uncover the specific yet collaborative contributions of each domain to the PHF7 ubiquitination activity.