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.

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.

SARS-CoV-2 ORF10 hijacking ubiquitination machinery reveals potential unique drug targeting sites

Viruses often manipulate ubiquitination pathways to facilitate their replication and pathogenesis.CUL2ZYG11B known as the substrate receptor of cullin-2 RING E3 ligase,is bound by SARS-CoV-2 ORF10 to increase its E3 ligase activity,leading to degradation of IFT46,a protein component of the intraflagellar transport(IFT)complex B.This results in dysfunctional cilia,which explains certain symptoms that are specific to COVID-19.However,the precise molecular mechanism of how ORF10 recognizes CUL2ZYG11B remains unknown.Here,we determined the crystal structure of CUL2ZYG11B complexed with the N-terminal extension(NTE)of SARS-CoV-2 ORF10(2.9 A°).The structure reveals that the ORF10 N-terminal heptapeptide(NTH)mimics the Gly/N-degron to bind CUL2ZYG11B.Mutagenesis studies identified key residues within ORF10 that are key players in its interaction with CUL2ZYG11B both in ITC assay and in vivo cells.In addition,we prove that enhancement of CUL2ZYG11B activity for IFT46 degradation by which ORF10-mediated correlates with the binding affinity between ORF10 and CUL2ZYG11B.Finally,we used a Global Protein Stability system to show that the NTH of ORF10 mimics the Gly/N-degron motif,thereby binding competitively to CUL2ZYG11B and inhibiting the degradation of target substrates bearing the Gly/N-degron motif.Overall,this study sheds light on how SARS-CoV-2 ORF10 exploits the ubiquitination machinery for proteasomal degradation,and offers valuable insights for optimizing PROTAC-based drug design based on NTH CUL2ZYG11B interaction,while pinpointing a promising target for the development of treatments for COVID-19.