Really Interesting New Gene Finger Protein Research Articles

Membrane-Associated Ubiquitin Ligase RING Finger Protein 152 Orchestrates Melanogenesis via Tyrosinase Ubiquitination.

Lysosomal degradation of tyrosinase, a pivotal enzyme in melanin synthesis, negatively impacts melanogenesis in melanocytes. Nevertheless, the precise molecular mechanisms by which lysosomes target tyrosinase have remained elusive. Here, we identify RING (Really Interesting New Gene) finger protein 152 (RNF152) as a membrane-associated ubiquitin ligase specifically targeting tyrosinase for the first time, utilizing AlphaScreen technology. We observed that modulating RNF152 levels in B16 cells, either via overexpression or siRNA knockdown, resulted in decreased or increased levels of both tyrosinase and melanin, respectively. Notably, RNF152 and tyrosinase co-localized at the trans-Golgi network (TGN). However, upon treatment with lysosomal inhibitors, both proteins appeared in the lysosomes, indicating that tyrosinase undergoes RNF152-mediated lysosomal degradation. Through ubiquitination assays, we found the indispensable roles of both the RING and transmembrane (TM) domains of RNF152 in facilitating tyrosinase ubiquitination. In summary, our findings underscore RNF152 as a tyrosinase-specific ubiquitin ligase essential for regulating melanogenesis in melanocytes.

Uncovering the mechanisms of MuRF1-induced ubiquitylation and revealing similarities with MuRF2 and MuRF3

MuRF1 (Muscle-specific RING finger protein 1; gene name TRIM63) is a ubiquitin E3 ligase, associated with the progression of muscle atrophy. As a RING (Really Interesting New Gene) type E3 ligase, its unique activity of ubiquitylation is driven by a specific interaction with a UBE2 (ubiquitin conjugating enzyme). Our understanding of MuRF1 function remains unclear as candidate UBE2s have not been fully elucidated. In the present study, we screened human ubiquitin dependent UBE2s in vitro and found that MuRF1 engages in ubiquitylation with UBE2D, UBE2E, UBE2N/V families and UBE2W. MuRF1 can cause mono-ubiquitylation, K48- and K63-linked polyubiquitin chains in a UBE2 dependent manner. Moreover, we identified a two-step UBE2 dependent mechanism whereby MuRF1 is monoubiquitylated by UBE2W which acts as an anchor for UBE2N/V to generate polyubiquitin chains. With the in vitro ubiquitylation assay, we also found that MuRF2 and MuRF3 not only share the same UBE2 partners as MuRF1 but can also directly ubiquitylate the same substrates: Titin (A168-A170), Desmin, and MYLPF (Myosin Light Chain, Phosphorylatable, Fast Skeletal Muscle; also called Myosin Light Regulatory Chain 2). In summary, our work presents new insights into the mechanisms that underpin MuRF1 activity and reveals overlap in MuRF-induced ubiquitylation which could explain their partial redundancy in vivo.

Mrz1, a Novel Mitochondrial Outer Membrane RING Finger Protein, is Degraded Through the Ubiquitin–Proteasome Pathway in Schizosaccharomyces pombe

The RING (Really Interesting New Gene) finger proteins are a large diverse group of Zinc finger proteins. Many determined RING finger proteins are ubiquitin-protein E3 ligases and RING E3s are the most abundant type of ubiquitin ligase. RING finger and RING finger E3s have been discovered in many organisms where they play various functions, including DNA repair, ubiquitination and mitochondrial protein quality control. In this study, we identified a novel mitochondrial protein (SPBC16G5.03)with predicted RING finger domain within an N-terminal 21-60 amino acids and named it Mrz1 (mitochondrial RING finger protein). Our results showed that Mrz1 is localized in the mitochondrial outer membrane. Deletion of mrz1 did not affect cell growth in an unstressed state, but increases sensitivity to selenite. We showed that Mrz1 was degraded during the stationary phase and blocked by addition proteasome inhibitor MG132. We further showed that the E2 enzyme Ubc13 was identified among 8 candidate proteins as the ubiquitin-conjugating enzyme in this system. These data suggested that the Mrz1 was degraded likely through the ubiquitin-proteasome system.

When RING finger family proteins meet SARS-CoV-2.

The pandemic coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is currently the most formidable challenge to humankind. Understanding the complicated virus-host interplay is crucial for fighting against viral infection. A growing number of studies point to the critical roles of RING (really interesting new gene) finger (RNF) proteins during SARS-CoV-2 infection. RNF proteins exert direct antiviral activity by targeting genome and envelope glycoproteins of SARS-CoV-2. Additionally, some RNF members serve as potent regulators for antiviral innate immunity and antibody-dependent neutralization of SARS-CoV-2. Notably, SARS-CoV-2 also hijacks the RNF proteins-mediated ubiquitination process to evade host antiviral innate immunity and enhance viral replication. In this mini-review, we discuss the diverse antiviral mechanisms of RNF proteins and viral immune evasion in an RNF proteins-dependent manner. Understanding the crosstalk between RNF proteins and SARS-CoV-2 infection would help design potential novel targets for COVID-19 treatment.

A New RING Finger Protein, PLANT ARCHITECTURE and GRAIN NUMBER 1, Affects Plant Architecture and Grain Yield in Rice.

Developing methods for increasing the biomass and improving the plant architecture is important for crop improvement. We herein describe a gene belonging to the RING_Ubox (RING (Really Interesting New Gene) finger domain and U-box domain) superfamily, PLANT ARCHITECTURE and GRAIN NUMBER 1 (PAGN1), which regulates the number of grains per panicle, the plant height, and the number of tillers. We used the CRISPR/Cas9 system to introduce loss-of-function mutations to OsPAGN1. Compared with the control plants, the resulting pagn1 mutant plants had a higher grain yield because of increases in the plant height and in the number of tillers and grains per panicle. Thus, OsPAGN1 may be useful for the genetic improvement of plant architecture and yield. An examination of evolutionary relationships revealed that OsPAGN1 is highly conserved in rice. We demonstrated that OsPAGN1 can interact directly with OsCNR10 (CELL NUMBER REGULATOR10), which negatively regulates the number of rice grains per panicle. A transcriptome analysis indicated that silencing OsPAGN1 affects the levels of active cytokinins in rice. Therefore, our findings have clarified the OsPAGN1 functions related to rice growth and grain development.

Oryza sativa, C4HC3-type really interesting new gene (RING), OsRFPv6, is a positive regulator in response to salt stress by regulating Na+ absorption.

Salinity negatively affects plant growth, productivity, and metabolism. Therefore, plants have evolved diverse strategies to survive in saline environments. To identify such strategies involving the ubiquitin/26S proteasome system, we characterized molecular functions of a rice C4HC3 really interesting new gene (RING)-type E3-ubiquitin ligase gene. Oryza sativa RING finger protein v6 (OsRFPv6) was highly expressed under conditions of abiotic stress, induced by 100 mM NaCl and 20% PEG. The GFP-OsRFPv6 protein was localized in the plasma membrane and cytosol in rice protoplasts. In vitro ubiquitin assay revealed that OsRFPv6 possessed E3-ubiquitin ligase activity, but its variant OsRFPv6C100A did not. OsRFPv6-overexpressing plants were insensitive to salinity, but their growth was delayed under normal conditions. Under saline conditions, transgenic plants exhibited higher proline, soluble sugar, and chlorophyll content and lower H2 O2 accumulation than wild-type plants. Moreover, transgenic plants exhibited lower Na+ uptake, lower Na+ content, and higher K+ content in the xylem sap assay. Under saline conditions, the expression levels of nine Na+ /K+ transporter genes in roots and leaves were significantly different between transgenic and wild-type plants. Specifically, under both normal and saline conditions, the expression of OsHKT2;1, a Na+ transporter, in the roots of transgenic plants was lower than that in the roots of wild-type plants. These results suggest that OsRFPv6 E3-ubiquitin ligase serves as a positive regulator of salinity response via Na+ uptake.

Crystal structure and mutational analysis of the human TRIM7 B30.2 domain provide insights into the molecular basis of its binding to glycogenin-1

Tripartite motif (TRIM)7 is an E3 ubiquitin ligase that was first identified through its interaction with glycogenin-1 (GN1), the autoglucosyltransferase that initiates glycogen biosynthesis. A growing body of evidence indicates that TRIM7 plays an important role in cancer development, viral pathogenesis, and atherosclerosis and, thus, represents a potential therapeutic target. TRIM family proteins share a multidomain architecture with a conserved N-terminal TRIM and a variable C-terminal domain. Human TRIM7 contains the canonical TRIM motif and a B30.2 domain at the C terminus. To contribute to the understanding of the mechanism of action of TRIM7, we solved the X-ray crystal structure of its B30.2 domain (TRIM7B30.2) in two crystal forms at resolutions of 1.6 Å and 1.8 Å. TRIM7B30.2 exhibits the typical B30.2 domain fold, consisting of two antiparallel β-sheets of seven and six strands, arranged as a distorted β-sandwich. Furthermore, two long loops partially cover the concave face of the β-sandwich defined by the β-sheet of six strands, thus forming a positively charged cavity. We used sequence conservation and mutational analyses to provide evidence of a putative binding interface for GN1. These studies showed that Leu423, Ser499, and Cys501 of TRIM7B30.2 and the C-terminal 33 amino acids of GN1 are critical for this binding interaction. Molecular dynamics simulations also revealed that hydrogen bond and hydrophobic interactions play a major role in the stability of a modeled TRIM7B30.2-GN1 C-terminal peptide complex. These data provide useful information that could be used to target this interaction for the development of potential therapeutic agents.

Molecular characterization of a RING E3 ligase SbHCI1 in sorghum under heat and abscisic acid stress.

Molecular function ofRING E3 ligase SbHCI1is involved in ABA-mediated basal heat stress tolerancein sorghum. Global warming generally reduces plant survival, owing to the negative effects of high temperatures on plant development. However, little is known about the role of Really Interesting New Gene (RING) E3 ligase in the heat stress responses of plants. As such, the aim of the present study was to characterize the molecular functions of the Sorghum bicolor ortholog of the Oryza sativa gene for Heat- and Cold-Induced RING finger protein 1 (SbHCI1). Subcellular localization revealed that SbHCI1 was mainly associated with the cytosol and that it moved to the Golgi apparatus under heat stress conditions. The fluorescent signals of SbHCI1 substrate proteins were observed to migrate to the cytoplasm under heat stress conditions. Bimolecular fluorescence complementation (BiFC) and yeast two-hybrid (Y2H) assays revealed that SbHCI1 physically interacted with OsHCI1 ortholog partner proteins in the cytoplasm. Moreover, an in vitro ubiquitination assay revealed that SbHCI1 polyubiquitinated each of the three interacting proteins. The ectopic overexpression of SbHCI1 in Arabidopsis revealed that the protein was capable of inducing abscisic acid (ABA)-hypersensitivity and basal heat stress tolerance. Therefore, SbHCI1 possesses E3 ligase activity and may function as a positive regulator of heat stress responses through the modulation of interacting proteins.

Molecular dissection of two homoeologous wheat genes encoding RING H2-type E3 ligases: TaSIRFP-3A and TaSIRFP-3B.

Two homoeologous wheat genes, TaSIRFP-3A and TaSIRFP-3B, encode the RING-HC-type E3 ligases that play an inhibitory role in sucrose metabolism in response to cold stress. In higher plants, the attachment of ubiquitin (Ub) and the subsequent recognition and degradation by the 26S proteasome affects a variety of cellular functions that are essential for survival. Here, we characterized the two homoeologous wheat genes encoding the really interesting new gene (RING) HC-type E3 ligases: TaSIRFP-3A and TaSIRFP-3B (Triticum aestivum SINA domain including RING finger protein 1 and 2), which regulate target proteins via the Ub/26S proteasome system. The TaSIRFP-3A gene was highly expressed under cold stress. In contrast, its homoeologous gene, TaSIRFP-3B, showed only a slight increase in expression levels in shoots. Despite these differences, both the proteins exhibited E3 ligase activity with the cytosol- and nucleus-targeted localization, demonstrating their conserved molecular function. Heterogeneous overexpression of TaSIRFP-3A or TaSIRFP-3B in Arabidopsis showed delayed plant growth causing a reduction in sucrose synthase enzymatic activity and photosynthetic sucrose synthesis, by regulating sucrose synthase proteins. TaSIRFP-3A- or TaSIRFP-3B-overexpressing plants showed higher hypersensitivity under cold stress than WT plants with an accumulation of reactive oxygen species (ROS). These results suggest that the negative regulation of TaSIRFP-3A and TaSIRFP-3B in response to cold stress is involved in sucrose metabolism.

A novel RING finger protein CqRNF152-like with self-ubiquitination activity inhibits white spot syndrome virus infection in a crustacean Cherax quadricarinatus

Really Interesting New Gene (RING) finger proteins are highly conserved molecules that participate in a variety of biological processes such as regulation of development, apoptosis and antiviral immunity in vertebrates. However, the functions of RING finger proteins are still poorly understood in crustaceans. Previously, we found that the transcript of a homolog of RING finger protein 152 (CqRNF152-like) was up-regulated in a differentially expressed transcriptome library of the haematopietic tissue (Hpt) cells from red claw crayfish Cherax quadricarinatus upon white spot syndrome virus (WSSV) infection, which is one of the most devastating viral diseases for crustaceans like shrimp and crayfish. The full-length cDNA sequence of CqRNF152-like was then identified with 975 bp, including an ORF of 685 bp that encoded a 195 amino acids protein, a 5′- UTR of 180 bp, and a 3′-UTR with a poly (A) tail of 207 bp. The conserved domain prediction showed that CqRNF152-like contained a conserved RING-finger domain. Gene expression analysis showed that CqRNF152-like was distributed in all tissues examined and the transcript is significantly up-regulated after WSSV challenge both in vivo in Hpt tissue and in vitro in cultured Hpt cells. Furthermore, the transcripts of both an immediate early gene ie1 and a late envelope protein gene vp28 of WSSV were clearly increased in the Hpt tissues, hemocytes and cultured Hpt cells after gene silencing of CqRNF152-like, which were further proved to be significantly decreased after overloading of recombinant CqRNF152-like protein in Hpt cell cultures. Meanwhile, CqRNF152-like was found to bind with WSSV envelope protein VP28 by proteins pull-down assay. Similar to most of RNF proteins, CqRNF152-like protein sequence contained a conserved RING-finger domain and showed self-ubiquitination activity in a RING finger domain dependent manner. Taken together, CqRNF152-like is likely to function as an antiviral molecular against WSSV infection through interaction with the envelope protein VP28 in a crustacean C. quadricarinatus. This is the first report that a RING finger protein with directly antiviral functions via interaction with viral protein and self-ubiquitination activity in crustacean, which sheds new light on the molecular mechanism of WSSV infection and the control of white spot disease.

Molecular Functions of Rice Cytosol-Localized RING Finger Protein 1 in Response to Salt and Drought and Comparative Analysis of Its Grass Orthologs.

In higher plants, the post-translational modification of target proteins via the attachment of molecules such as ubiquitin (Ub) mediates a variety of cellular functions via the Ub/26S proteasome system. Here, a really interesting new gene (RING)-H2 type E3 ligase, which regulates target proteins via the Ub/26S proteasome system, was isolated from a rice plant, and its other grass orthologs were examined to determine the evolution of its molecular function during speciation. The gene encoding Oryza sativa cytoplasmic-localized RING finger protein 1 (OsCLR1) was highly expressed under salt and drought stresses. By contrast, the three grass orthologs, SbCLR1 from Sorghum bicolor, ZmCLR1 from Zea mays and TaCLR1 from Triticum aestivum, showed different responses to these stresses. Despite these differences, all four orthologs exhibited E3 ligase activity with cytosol-targeted localization, demonstrating conserved molecular functions. Although OsCLR1-overexpressing plants showed higher survival rates under both salt and drought stresses than that of the wild type (WT) plants, this pattern was not observed in the other orthologs. In addition, OsCLR1-overexpressing plants exhibited lower germination rates in ABA than that of WT plants, whereas the three ortholog CLR1-overexpressing plants showed rates similar to the WT plants. These results indicate the positive regulation of OsCLR1 in response to salt and drought in an ABA-dependent manner. Despite the molecular functions of the three CLR1 orthologs remaining largely unknown, our results provide an insight into the evolutionary fate of CLR1 grass orthologs during speciation after the divergence from a common ancestor.

Methods to analyze STUbL activity.

Posttranslational modification with small ubiquitin-like modifier (SUMO) plays an important role in many biological processes. SUMO-targeted ubiquitin E3 ligases (STUbLs) are part of the really interesting new gene (RING)-type family of ubiquitin E3 ligases. STUbLs recognize their SUMO-modified substrates via SUMO-interaction motifs and ubiquitinate them via the RING domain. As a result, they form a link between the ubiquitin and SUMO signaling pathways. STUbL activity is required for the maintenance of genome stability, the repair of damaged DNA and to target SUMO-modified proteins for degradation by the proteasome. In vitro assays for STUbL activity have been developed and used to identify their cognate ubiquitin-conjugating enzymes (E2s), to determine their substrate requirements, and to characterize the types of ubiquitin chains linked to substrates. While we have focused on the STUbL RING finger protein 4 (RNF4) the methods we describe can be extended to other STUbLs. We also describe an assay for RNF4 ubiquitination activity based on fluorescence polarization, suitable for high-throughput compound screening in drug discovery.

Repertoire of plant RING E3 ubiquitin ligases revisited: New groups counting gene families and single genes.

E3 ubiquitin ligases of the ubiquitin proteasome system (UPS) mediate recognition of substrates and later transfer the ubiquitin (Ub). They are the most expanded components of the system. The Really Interesting New Gene (RING) domain contains 40–60 residues that are highly represented among E3 ubiquitin ligases. The Arabidopsis thaliana E3 ubiquitin ligases with a RING finger primarily contain RING-HC or RING-H2 type domains or less frequently RING-v, RING-C2, RING-D, RING-S/T and RING-G type domains. Our previous work on three E3 ubiquitin ligase families with a RING-H2 type domain, ATL, BTL, and CTL, suggested that a phylogenetic distribution based on the RING domain allowed for the creation a catalog of known domains or unknown conserved motifs. This work provided a useful and comprehensive view of particular families of RING E3 ubiquitin ligases. We updated the annotation of A. thaliana RING proteins and surveyed RING proteins from 30 species across eukaryotes. Based on domain architecture profile of the A. thaliana proteins, we catalogued 4711 RING finger proteins into 107 groups, including 66 previously described gene families or single genes and 36 novel families or undescribed genes. Forty-four groups were specific to a plant lineage while 41 groups consisted of proteins found in all eukaryotic species. Our present study updates the current classification of plant RING finger proteins and reiterates the importance of these proteins in plant growth and adaptation.

A Negative Regulator in Response to Salinity in Rice: Oryza sativa Salt-, ABA- and Drought-Induced RING Finger Protein 1 (OsSADR1).

RING (Really Interesting New Gene) finger proteins play crucial roles in abiotic stress responses in plants. We report the RING finger E3 ligase gene, an Oryza sativa salt, ABA and drought stress-induced RING finger protein 1 gene (OsSADR1). We demonstrated that although OsSAR1 possesses E3 ligase activity, a single amino acid substitution (OsSADR1C168A) in the RING domain resulted in no E3 ligase activity, suggesting that the activity of most E3s is specified by the RING domain. Additional assays substantiated that OsSADR1 interacts with three substrates-no E3 ligase acti and OsPIRIN, and mediates their proteolysis via the 26S proteasome pathway. For OsSADR1, approximately 62% of the transient signals were in the cytosol and 38% in the nucleus. However, transiently expressed OsSADR1 was primarily expressed in the nucleus (70%) in 200 mM salt-treated rice protoplasts. The two nucleus-localized proteins (OsSNAC2 and OsGRAS44) interacted with OsSADR1 in the cytosol and nucleus. Heterogeneous overexpression of OsSADR1 in Arabidopsis resulted in sensitive phenotypes for salt- and mannitol-responsive seed germination and seedling growth. With ABA, OsSADR1 overexpression in plants produced highly tolerant phenotypes, with morphological changes in root length and stomatal closure. The ABA-tolerant transgenic plants also showed hypersensitivity phenotypes under severe water deficit conditions. Taken together, OsSADR1 may act as a regulator in abiotic stress responses by modulating target protein levels.

Neuroprotective effect of really interesting novel gene finger protein 146/Iduna and its pro-survival activity in disease

Background The really interesting new gene (RING) finger protein 146/Iduna is a kind of endogenous pro-survival protein and can combine with poly ADP-ribose (PAR) to effectively interfere the PARP1-dependent nerve cell parthanatos death caused by excitotoxicity and oxidative stress. Some researches also found that Iduna had the E3 ubiquitin ligase activity and participated in the DNA repair, which played the regulating effect in many diseases through the Wnt pathway. Objective To understand the biological nature of RING finger 146/Iduna protein and its application in diseases. Content Review the neuroprotective effect and possible mechanism of endogenous pro-survival activity in different diseases and cell damage models of RING finger protein 146/Iduna. Trend With the further research on the biological function of RING finger protein 146/Iduna, its role in the disease will be revealed constantly. Key words: Really interesting new gene finger protein 146; Iduna; Neuroprotective; Pro-survival activity

RING finger protein 113A regulates C-X-C chemokine receptor type 4 stability and signaling.

As an α-chemokine receptor specific for stromal-derived-factor-1 (SDF-1, also called CXCL12), C-X-C chemokine receptor type 4 (CXCR4) plays a vital role in chemotactically attracting lymphocytes during inflammation. CXCR4 also regulates HIV infection due to its role as one of the chemokine coreceptors for HIV entry into CD4+ T cells. Chemokine receptors and their signaling pathways have been shown to be regulated by the process of ubiquitination, a posttranslational modification, guided by ubiquitin E3 ligases, which covalently links ubiquitin chains to lysine residues within target substrates. Here we describe a novel mechanism regulating CXCR4 protein levels and subsequent CXCR4/CXCL12 signaling pathway through the ubiquitination and degradation of the receptor in response to ligand stimulation. We identify that an uncharacterized really interesting new gene (RING) finger ubiquitin E3 ligase, RING finger protein 113A (RNF113A), directly ubiquitinates CXCR4 in cells, leading to CXCR4 degradation, and therefore disrupts the signaling cascade. We determined that the K331 residue within CXCR4 is essential for RNF113A-mediated ubiquitin conjugation. Overexpression of RNF113A significantly reduces CXCL12-induced kinase activation in HeLa cells, whereas RNF113A knockdown enhances CXCL12-induced downstream signaling. Further, RNF113A expression and silencing directly affect cell motility in a wound healing assay. These results suggest that RNF113A plays an important role in CXCR4 signaling through the ubiquitination and degradation of CXCR4. This mechanistic study might provide new understanding of HIV immunity and neutrophil activation and motility regulated by CXCR4.

RING Finger Protein 38 Is a Neuronal Protein in the Brain of Nile Tilapia, Oreochromis niloticus.

Really interesting new gene (RING) finger protein is a type of zinc-binding motif found in a large family of functionally distinct proteins. RING finger proteins are involved in diverse cellular processes including apoptosis, DNA repair, cell cycle, signal transduction, tumour suppressor, vesicular transport, and peroxisomal biogenesis. RING finger protein 38 (RNF38) is a member of the family whose functions remain unknown. To gain insight into the putative effects of RNF38 in the central nervous system, we localised its expression. The aim of this study was to identify the neuroanatomical location(s) of rnf38 mRNA and its peptide, determine the type of RNF38-expressing cells, and measure rnf38 gene expression in the brain of male tilapia. The distributions of rnf38 mRNA and its peptide were visualised using in situ hybridisation with digoxigenin-labelled RNA antisense and immunocytochemistry, respectively. Both were identically distributed throughout the brain, including the telencephalon, preoptic area, optic tectum, hypothalamus, cerebellum, and the hindbrain. Double-labelling immunocytochemistry for RNF38 and the neuronal marker HuC/D showed that most but not all RNF38 protein was expressed in neuronal nuclei. Quantitative real-time polymerase chain reaction showed the highest level of rnf38 mRNA in the midbrain, followed by the preoptic area, cerebellum, optic tectum, telencephalon, hindbrain and hypothalamus. These findings reveal a differential spatial pattern of RNF38 in the tilapia brain, suggesting that it has potentially diverse functions related to neuronal activity.

Structure and Function of the RING Domains of RNF20 and RNF40, Dimeric E3 Ligases that Monoubiquitylate Histone H2B

Monoubiquitylation of histone H2B is a post-translational mark that plays key roles in regulation of transcription and genome stability. In humans, attachment of ubiquitin to lysine 120 of histone H2B depends on the activity of the E2 ubiquitin-conjugating enzyme, Ube2B, and the really interesting new gene (RING) E3 ligases, RING finger protein (RNF) 20 and RNF40. To better understand the molecular basis of this modification, we have solved the crystal structure of the RNF20 RING domain and show that it is a homodimer that specifically interacts with the Ube2B~Ub conjugate. By mutating residues at the E3–E2 and E3–ubiquitin interfaces, we identify key contacts required for interaction of the RNF20 RING domain with the Ube2B~Ub conjugate. These mutants were used to generate a structure-based model of the RNF20–Ube2B~Ub complex that reveals differences from other RING-E2~Ub complexes, and suggests how the RNF20–Ube2B~Ub complex might interact with its nucleosomal substrate. Additionally, we show that the RING domains of RNF20 and RNF40 can form a stable heterodimer that is active. Together, our studies provide new insights into the mechanisms that regulate RNF20-mediated ubiquitin transfer from Ube2B.

Molecular dissection of Oryza sativa salt-induced RING Finger Protein 1 (OsSIRP1): possible involvement in the sensitivity response to salinity stress.

Ubiquitination-mediated protein degradation via Really Interesting New Gene (RING) E3 ligase plays an important role in plant responses to abiotic stress conditions. Many plant studies have found that RING proteins regulate the perception of various abiotic stresses and signal transduction. In this study, Oryza sativa salt-induced RING Finger Protein 1 (OsSIRP1) gene was selected randomly from 44 Oryza sativa RING Finger Proteins (OsRFPs) genes highly expressed in rice roots exposed to salinity stress. Transcript levels of OsSIRP1 in rice leaves after various stress treatments, including salt, heat, drought and hormone abscisic acid (ABA), were observed. Poly-ubiquitinated products of OsSIRP1 were investigated via an in vitro ubiquitination assay.35S:OsSIRP1-EYFP was distributed in the cytosol of untreated and salt-treated rice protoplasts. Heterogeneous overexpression of OsSIRP1 in Arabidopsis reduced tolerance for salinity stress during seed germination and root growth. Our findings indicate that OsSIRP1 acts as a negative regulator of salinity stress tolerance mediated by the ubiquitin 26S proteasome system.

Cloning and serotonergic regulation of RING finger protein38 (rnf38) in the brain of medaka (Oryzias latipes)

Serotonin (5-HT) is a key regulator of mood and sexual behaviors. 5-HT reuptake inhibitors have been used as antidepressants. Really interesting new gene (RING) finger proteins have been associated with 5-HT regulation but their role remains largely unknown. Some RING finger proteins are involved in the serotonergic system, therefore, we speculate that the gene expression of RING finger protein38 (rnf38) is regulated by the serotonergic system. In the present study, we aimed to identify the full length sequence of medaka (Oryzias latipes) rnf38 mRNA and investigate its association with the serotonergic system using an antidepressant, citalopram (CIT). We identified the full length rnf38 cDNA, which consisted of 2726 nucleotides spanning 12 exons and the deduced protein sequence consisting of 518 amino acid residues including a RING finger domain, a KIT motif and a coiled-coil domain. Medaka exposed to 10−7M of CIT showed anxiety-like behavior. The expressions of 5-HT-related genes, pet1, solute carrier family 6, member 4A (slc6a4) and tryptophan hydroxylase (tph2) were significantly low (P<0.05) in the hindbrain. On the other hand, rnf38 gene was significantly high (P<0.05) in the telencephalon and the hypothalamus. This shows that 5-HT synthesis and transport in the hindbrain is suppressed by CIT, which induces rnf38 gene expression in the forebrain where 5-HT neurons project. Thus, the expression of rnf38 is negatively regulated by the serotonergic system.