The T-cell receptor (TCR) engages with an antigen and initiates a signaling cascade that leads to the activation of transcription factors. Roquin, a protein encoded by the RC- 3H1 gene and characterized as an immune regulator, was recently identified as a novel RING-type ubiquitin ligase family member, but the mechanisms by which Roquin regulates T-cell responses are unclear. We used the EL-4 murine lymphoma cell line to elucidate the role of Roquin in vitro. Roquin-overexpressing EL-4 cells became hyper-responsive after anti-CD3/CD28 stimulation in vitro and were a major source of the cytokines IL-2 and TNF-α. Upon activation, these cells showed particularly enhanced production of IL-2 and TNF-α. To clarify the important role played by Roquin in T-cell responses ex vivo, we generated T-cell-specific Roquin transgenic (Tg) mice. Roquin-Tg CD4+ T-cells showed enhanced production of IL-2 and TNF-α in response to TCR stimulation with anti-CD28 co-stimulation. Further studies are necessary to investigate the role of Roquin in the regulation of primary T-cell activation, survival, and differentiation.

As sessile organisms, plants have evolved mechanisms that allow them to adapt and survive periods of various environmental stresses including high salinity and drought. The ubiquitin-proteasome system (UPS) is an integral player in plant response and adaptation to various abiotic stresses. Understanding UPS function has centered mainly on defining the role of E3 ubiquitin ligases, which are the substrate-recruiting component of the ubiquitination pathway. Here, we report on Ring finger E3 ligase, Oryza sativa salt- and drought-induced RING finger protein1 gene (OsSDRFP1) in defense responses to osmotic stresses. Results of qRT-PCR and In vitro ubiquitination assay demonstrated that OsSDRFP1 act as an E3 ligase in response to salt and drought stresses. in this study, Subcellular localizations showed that the OsSDRFP1 was observed in cytosol (66%) and nucleus (34%) under non-treated conditions. However, the florescence signals of rice protoplasts after salt treatments detected in nucleus (60%) higher than in cytosol (30%). The Arabidopsis plants overexpressing OsSDRFP1 clearly exhibited hypersensitive responses to salt stress. whereas, OsSDRFP1-overexpressing plants were more tolerant to both drought- and ABA-stresses than the wild-type plants. These results might suggest that OsSDRFP1 has a dual function as a regulator of high salt- and drought-stresses.

The metalloid arsenic (As) and the hevy metal cadmium (Cd) are ubiquitously found at low concentrations in the earth, while high concentrations of the both elements in soil and crop are severe dangerous to human health. We have tried to retrieve RING E3 ligase gene, which is believed to regulate substrate proteins in As or Cd uptake via ubiquitin 26S proteasome pathway, related to inhibit metal ion transport system. A total of 48 rice RING E3 ligases were randomly selected and then conducted semi-quantitative RT-PCR for their expression patterns as exposed to As and Cd treatments. We discovered one gene, Oryza sativa heavy metal induced RING E3 ligase 1 (OsHIR1) that was significantly up-regulated against both treatments. A total of 31 positive interaction clones with OsHIR1 were screened depending on their strong α-galactosidase activity via yeast-two hybrid screen. Bimolecular fluorescence complementation analysis evidenced that the OsHIR1 protein was clearly interacted with each of six partner protein including aquaporin tonoplast intrinsic protein 4;1 (OsTIP4;1) in the plasma membrane. Protein degradation assay showed that OsHIR1 strongly degraded the protein level of OsTIP4;1 via ubiquitin 26S proteasome system. Heterogeneous overexpression of OsHIR1 in Arabidopsis showed As- and Cd-insensitive phenotype. In addition, the transgenic plant showed low levels of As and Cd accumulation than the control plant in leaf and root. Here, we report the novel finding that OsHIR1 E3 ligase positively regulates OsTIP4;1 related to As and Cd uptake.

Plant growth under water-deficit conditions adversely affects many key processes. Efforts to understand drought stress-related defense mechanisms have revealed a host of plant genes using molecular approaches in rice. Here, we report the novel finding that OsCTR1 E3 ligase regulates both chloroplast-localized chloroplast protein 12 (OsCP12) and ribosomal protein 1 (OsRP1) in protein levels and subcellular localization. The results of a yeast-two hybrid assay, bimolecular fluorescence complementation assay, ubiquitination assay, subcellular localization, and a protein degradation assay support the hypothesis that OsCTR1 functions in trafficking inhibition and proteolysis of OsCP12 and OsRP1 via the ubiquitin 26S proteasome pathway. Heterogeneous overexpression of OsCTR1 in Arabidopsis showed ABA-hypersensitive phenotype in seed germination, seedling growth, and stomatal closure. The transgenic plants also exhibited improvement of water-deficit tolerance with an accumulation of hydrogen peroxide production. These results demonstrate that the OsCTR1 E3 ligase might positively regulate the cellular functions of OsCP12 and RP1 related to photosynthesis under drought stress conditions in rice.

Plants are known to have homeostatic cellular mechanisms to control the concentration of heavy metal inside the cell. We tried to retrieve rice RING finger protein genes, which are believed to regulate substrates via ubiqitinations, related to metal ions detoxification mechanisms. A total of 48 rice RING finger proteins were randomly selected and then examined for their expression patterns as exposed to cadmium and arsenic treatments. We discovered a RING finger protein gene that was significant up-regulated against both treatments and then named Oryza sativa heavy metal induced 1 (OsHMI1). We tested subsequently OsHMI1 expression patterns against to salinity, dehydration, cold, heat stress and phytohormones treatments. In addition, we evaluated its subcellular localization and determined E3 ligase activity. The interaction partner proteins were screened via yeast-two hybridization. These results might shed further light on the understanding of homeostatic cellular mechanisms to control heavy metal detoxification via protein degradation in plants.