Disruption of zinc (II) binding and dimeric protein structure of the XIAP-RING domain by copper (I) ions.

Modulation of metalloprotein structure and function via metal ion substitution may constitute a molecular basis for metal ion toxicity and/or metal-mediated functional control. The X-linked Inhibitor of Apoptosis Protein (XIAP) is a metalloprotein that requires zinc for proper structure and function. In addition to its role as a modulator of apoptosis, XIAP has been implicated in copper homeostasis. Given the similar coordination preferences of copper and zinc, investigation of XIAP structure and function upon interaction with copper is relevant. The Really Interesting New Gene (RING) domain of XIAP is representative of a class of zinc finger proteins that utilize a bi-nuclear zinc-binding motif to maintain proper structure and ubiquitin ligase function. Herein, we report the characterization of copper (I) binding to the Zn2-RING domain of XIAP. Electronic absorption studies that monitor copper-thiolate interactions demonstrate that the RING domain of XIAP binds 5-6 Cu(I) ions and that copper is thermodynamically preferred relative to zinc. Repetition of the experiments in the presence of the Zn(II)-specific dye Mag-Fura2 shows that Cu(I) addition results in Zn(II) ejection from the protein, even in the presence of glutathione. Loss of dimeric structure of the RING domain, which is a requirement for its ubiquitin ligase activity, upon copper substitution at the zinc-binding sites, was readily observed via size exclusion chromatography. These results provide a molecular basis for the modulation of RING function by copper and add to the growing body of literature that describe the impact of Cu(I) on zinc metalloprotein structure and function.

Inactivation of the MDM2 RING domain enhances p53 transcriptional activity in mice.

The MDM2 RING domain harbors E3 ubiquitin ligase activity critical for regulating the degradation of tumor suppressor p53, which controls many cellular pathways. The MDM2 RING domain also is required for an interaction with MDMX. Mice containing a substitution in the MDM2 RING domain, MDM2C462A, disrupting MDM2 E3 function and the MDMX interaction, die during early embryogenesis that can be rescued by p53 deletion. To investigate whether MDM2C462A, which retains p53 binding, has p53-suppressing activity, we generated Mdm2C462A/C462A ;p53ER/- mice, in which we replaced the endogenous p53 alleles with an inducible p53ER/- allele, and compared survival with that of similarly generated Mdm2-/-;p53ER/- mice. Adult Mdm2-null mice died ∼7 days after tamoxifen-induced p53 activation, indicating that in the absence of MDM2, MDMX cannot suppress p53. Surprisingly, Mdm2C462A/C462A ;p53ER/- mice died ∼5 days after tamoxifen injection, suggesting that p53 activity is higher in the presence of MDM2C462A than in the absence of MDM2. Indeed, in MDM2C462A-expressing mouse tissues and embryonic fibroblasts, p53 exhibited higher transcriptional activity than in those expressing no MDM2 or no MDM2 and MDMX. This observation indicated that MDM2C462A not only is unable to suppress p53 but may have gained the ability to enhance p53 activity. We also found that p53 acetylation, a measure of p53 transcriptional activity, was higher in the presence of MDM2C462A than in the absence of MDM2. These results reveal an unexpected role of MDM2C462A in enhancing p53 activity and suggest the possibility that compounds targeting MDM2 RING domain function could produce even more robust p53 activation.

The RING domain of RAG1 ubiquitylates histone H3: a novel activity in chromatin-mediated regulation of V(D)J joining.

The RAG1 and RAG2 proteins are the only lymphoid-specific factors required to perform the first step of V(D)J recombination, DNA cleavage. While the catalytic domain of RAG1, the core region, has been well characterized, the role of the noncore region in modulating chromosomal V(D)J recombination efficiency remains ill defined. Recent studies have highlighted the role of chromatin structure in regulation of V(D)J recombination. Here we show that RAG1 itself, through a RING domain within its N-terminal noncore region, preferentially interacts directly with and promotes monoubiquitylation of histone H3. Mutations affecting the RAG1 RING domain reduce histone H3 monoubiquitylation activity, decrease V(D)J recombination activity in vivo, reduce formation of both signal-joint and coding-joint products on episomal substrates, and decrease efficiency of V(D)J recombination at the endogenous IgH locus in lymphoid cells. The results reveal that RAG1-mediated histone monoubiquitylation activity plays a role in regulating the joining phase of chromosomal V(D)J recombination.

The putative PRC1 RING-finger protein AtRING1A regulates flowering through repressing MADS AFFECTING FLOWERING genes in Arabidopsis.

Polycomb group proteins play essential roles in the epigenetic control of gene expression in plants and animals. Although some components of Polycomb repressive complex 1 (PRC1)-like complexes have recently been reported in the model plant Arabidopsis, how they contribute to gene repression remains largely unknown. Here we show that a putative PRC1 RING-finger protein, AtRING1A, plays a hitherto unknown role in mediating the transition from vegetative to reproductive development in Arabidopsis. Loss of function of AtRING1A results in the late-flowering phenotype, which is attributed to derepression of two floral repressors, MADS AFFECTING FLOWERING 4/5 (MAF4/5), which in turn downregulate two floral pathway integrators, FLOWERING LOCUS T and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1. Levels of the H3K27me3 repressive mark at MAF4 and MAF5 loci, which is deposited by CURLY LEAF (CLF)-containing PRC2-like complexes and bound by LIKE HETEROCHROMATIN PROTEIN 1 (LHP1), are affected by AtRING1A, which interacts with both CLF and LHP1. Levels of the H3K4me3 activation mark correlate inversely with H3K27me3 levels at MAF4 and MAF5 loci. Our results suggest that AtRING1A suppresses the expression of MAF4 and MAF5 through affecting H3K27me3 levels at these loci to regulate the floral transition in Arabidopsis.

Polyubiquitination of the demethylase Jhd2 controls histone methylation and gene expression.

The identification of histone methyltransferases and demethylases has uncovered a dynamic methylation system needed to modulate appropriate levels of gene expression. Gene expression levels of various histone demethylases, such as the JARID1 family, show distinct patterns of embryonic and adult expression and respond to different environmental cues, suggesting that histone demethylase protein levels must be tightly regulated for proper development. In our study, we show that the protein level of the yeast histone H3 Lys 4 (H3 K4) demethylase Jhd2/Kdm5 is modulated through polyubiquitination by the E3 ubiquitin ligase Not4 and turnover by the proteasome. We determine that polyubiquitin-mediated degradation of Jhd2 controls in vivo H3 K4 trimethylation and gene expression levels. Finally, we show that human NOT4 can polyubiquitinate human JARID1C/SMCX, a homolog of Jhd2, suggesting that this is likely a conserved mechanism. We propose that Not4 is an E3 ubiquitin ligase that monitors and controls a precise amount of Jhd2 protein so that the proper balance between histone demethylase and histone methyltransferase activities occur in the cell, ensuring appropriate levels of H3 K4 trimethylation and gene expression.

Amyloplast displacement is necessary for gravisensing in Arabidopsis shoots as revealed by a centrifuge microscope.

The starch-statolith hypothesis proposes that starch-filled amyloplasts act as statoliths in plant gravisensing, moving in response to the gravity vector and signaling its direction. However, recent studies suggest that amyloplasts show continuous, complex movements in Arabidopsis shoots, contradicting the idea of a so-called 'static' or 'settled' statolith. Here, we show that amyloplast movement underlies shoot gravisensing by using a custom-designed centrifuge microscope in combination with analysis of gravitropic mutants. The centrifuge microscope revealed that sedimentary movements of amyloplasts under hypergravity conditions are linearly correlated with gravitropic curvature in wild-type stems. We next analyzed the hypergravity response in the shoot gravitropism 2 (sgr2) mutant, which exhibits neither a shoot gravitropic response nor amyloplast sedimentation at 1 g. sgr2 mutants were able to sense and respond to gravity under 30 g conditions, during which the amyloplasts sedimented. These findings are consistent with amyloplast redistribution resulting from gravity-driven movements triggering shoot gravisensing. To further support this idea, we examined two additional gravitropic mutants, phosphoglucomutase (pgm) and sgr9, which show abnormal amyloplast distribution and reduced gravitropism at 1 g. We found that the correlation between hypergravity-induced amyloplast sedimentation and gravitropic curvature of these mutants was identical to that of wild-type plants. These observations suggest that Arabidopsis shoots have a gravisensing mechanism that linearly converts the number of amyloplasts that settle to the 'bottom' of the cell into gravitropic signals. Further, the restoration of the gravitropic response by hypergravity in the gravitropic mutants that we tested indicates that these lines probably have a functional gravisensing mechanism that is not triggered at 1 g.

Characterization of the gene structure, functional significance, and clinical application of RNF180, a novel gene in gastric cancer.

BACKGROUND: By using genome-wide methylation screening, the authors identified ring finger protein 180 (RNF180) as preferentially methylated in cancer. This study was undertaken to clarify its structure and functional role in gastric cancer. METHODS: The transcription start site and core functional promoter region of RNF180 were revealed by 5' rapid amplification of cDNA ends and luciferase activity assays. Promoter methylation was detected by combined bisulfite restriction analysis and bisulfite genomic sequencing. Cell growth was detected by colony formation assay, apoptosis by annexin V assay, and RNF180 target genes by cDNA microarray. RESULTS: The authors revealed the transcription start site of RNF180 gene and identified the functional core promoter region (-202/+372) in the CpG island, which could be silenced by in vitro methylation assay. RNF180 was silenced in 6 of 7 gastric cancer cell lines and significantly down-regulated in primary gastric cancers compared with adjacent normal tissues (P = .001). Loss of gene expression was associated with promoter methylation. Re-expression of RNF180 suppressed cell growth (P < .001) and induced apoptosis (P < .05), which were mediated by up-regulating the antiproliferation regulators MTSS1 and CDKN2A and the proapoptotic mediator TIMP3. Promoter methylation of RNF180 was detected in 76% (150 of 198) of primary gastric cancers and 55% (11 of 20) of intestinal metaplasia, but in none of 23 normal gastric tissues. Methylated RNF180 DNA was detected in the plasma of 56% of gastric cancer patients, but not in healthy controls (P = .003). Patients with low or loss of RNF180 expression had significantly poorer overall survival. CONCLUSIONS: RNF180 is a novel potential tumor suppressor in gastric carcinogenesis and has potential clinical utility as a biomarker for gastric cancer patients.

Oestrogen causes degradation of KLF5 by inducing the E3 ubiquitin ligase EFP in ER-positive breast cancer cells.

KLF5 (Krüppel-like factor 5) is a multifunctional transcription factor involved in cell proliferation, differentiation and carcinogenesis. In addition to frequent inactivation in different types of human cancers, including breast cancer, KLF5 has been identified as an essential co-factor for the TGF-ß (transforming growth factor ß) tumour suppressor. In our previous study demonstrating a negative regulation of ER (oestrogen receptor α) function by KLF5 in breast cancer cells [Guo, Dong, Zhao, Sun, Li and Dong (2010) Int. J. Cancer 126, 81-89], we noticed that oestrogen reduced the protein level of KLF5. In the present study, we have tested whether and how oestrogen/ER signalling regulates KLF5 protein. We found that oestrogen caused the degradation of KLF5 protein, and the degradation was sensitive to proteasome inhibitors, but not other inhibitors. The oestrogen-inducible E3 ligase EFP (oestrogen-responsive finger protein) was identified as a key player in oestrogen-mediated degradation of KLF5, as knockdown and overexpression of EFP increased and decreased KLF5 protein levels respectively, and the decrease continued even when protein synthesis was blocked. EFP-mediated degradation impaired the function of KLF5 in gene transcription. Although only unubiquitinated EFP interacted with KLF5, overexpression of EFP appeared to prevent the ubiquitination of KLF5, while resulting in heavy ubiquitination of the E3 itself. Furthermore, ubiquitination of EFP interrupted its interaction with KLF5. Although the mechanism for how EFP degrades KLF5 remains to be determined, the results of the present study suggest that oestrogen causes the degradation of KLF5 protein by inducing the expression of EFP in ER-positive breast cancer cells.

Cargo-selected transport from the mitochondria to peroxisomes is mediated by vesicular carriers.

Mitochondria and peroxisomes share a number of common biochemical processes, including the beta oxidation of fatty acids and the scavenging of peroxides. Here, we identify a new outer-membrane mitochondria-anchored protein ligase (MAPL) containing a really interesting new gene (RING)-finger domain. Overexpression of MAPL leads to mitochondrial fragmentation, indicating a regulatory function controlling mitochondrial morphology. In addition, confocal- and electron-microscopy studies of MAPL-YFP led to the observation that MAPL is also incorporated within unique, DRP1-independent, 70-100 nm diameter mitochondria-derived vesicles (MDVs). Importantly, vesicles containing MAPL exclude another outer-membrane marker, TOM20, and vesicles containing TOM20 exclude MAPL, indicating that MDVs selectively incorporate their cargo. We further demonstrate that MAPL-containing vesicles fuse with a subset of peroxisomes, marking the first evidence for a direct relationship between these two functionally related organelles. In contrast, a distinct vesicle population labeled with TOM20 does not fuse with peroxisomes, indicating that the incorporation of specific cargo is a primary determinant of MDV fate. These data are the first to identify MAPL, describe and characterize MDVs, and define a new intracellular transport route between mitochondria and peroxisomes.

Secretory protein with RING finger domain (SPRING) specific to Trypanosoma cruzi is directed, as a ubiquitin ligase related protein, to the nucleus of host cells.

While some intracellular bacterial and viral proteins secreted into host cell possess ubiquitin ligase (E3) activity for their profit, it has not been reported whether intracellular parasites secrete such molecules. We identified a gene that encodes a protein containing a secretory signal peptide and a RING finger domain in the intracellular protozoan parasite, Trypanosoma cruzi. This gene was specific to T. cruzi and was designated spring (secretory protein with RING finger domain). An in vitro ubiquitination assay showed that SPRING possessed E3 activity in a RING finger domain-dependent manner. SPRING could utilize human ubiquitin-activating enzymes (E2), UbcH5 and UbcH13. Although SPRING was found to be a secretory protein, the signal peptide-cleaved mature form of SPRING was localized in the nucleus of host cells, indicating that SPRING may function in the host cell nuclei. Yeast two-hybrid screening identified 52 putative SPRING interactors in HeLa cells, suggesting that SPRING affects the stability or function of a number of host proteins. Furthermore, a co-immunoprecipitation assay showed that breast cancer-associated protein 3 interacted with SPRING, as well as being ubiquitinated by SPRING in vitro. These findings are the first to show that this protozoan parasite secretes an ubiquitin ligase-related protein into host cells.

An important role of PHRF1 in dendritic architecture and memory formation by modulating TGF-ß signaling.

PHRF1 is involved in transforming growth factor ß (TGF-ß) signaling to constrain the formation of acute promyelocytic leukemia (APL) in mouse APL models. PHRF1 also participates in modulating non-homologous end-joining. However, the role of PHRF1 in mammalian dendrite architecture and synaptic plasticity is unclear. Here, we investigated the role of PHRF1 in dendritic formation in the murine hippocampus using Camk2a promoter driven-iCre recombinase to conduct a PHRF1 conditional knockout, namely PHRF1Δ/Δ, in the forebrain region. PHRF1Δ/Δ mice developed normally, but exhibited anxiety-like behaviors and displayed defective spatial memory. Alterations of dendritic complexity in apical and basal dendrites of pyramidal neurons were noticed in PHRF1Δ/Δ mutants. Furthermore, electrical stimulation in the hippocampal CA1 region after the TGF-ß1 treatment showed a reduced synaptic plasticity in PHRF1Δ/Δ mice. Immunoblotting analysis indicated that PHRF1 ablation affected the TGF-ß signaling. Collectively, our results demonstrate that PHRF1 is important for the dendritic architecture and required for spatial memory formation in the hippocampus.

SbRFP1 regulates cold-induced sweetening of potato tubers by inactivation of StBAM1.

Potato cold-induced sweetening (CIS) is a major drawback restricting potato process industry. Starch degradation and sucrose decomposition are considered to be the key pathways in potato CIS. Our previous study showed that the RING finger gene SbRFP1 could slow down starch degradation and the accumulation of reducing sugars (RS) through inhibiting amylase and invertase activity in cold-stored tubers. However, the regulation mechanism of SbRFP1 is not clear. In this paper, we first proved that SbRFP1 could promote starch synthesis and modify the shape of starch granules. By further yeast two hybrid, GST-pull down and inhibition of enzyme activity assays, we confirmed that SbRFP1 could slow down the transformation of starch to RS in tubers mainly through the inhibition of ß-amylase StBAM1 activity. SbRFP1 was also proved to possess E3 ubiquitin ligase activity by ubiquitination assay. Thus, SbRFP1 may regulate the accumulation of RS in cold-stored tubers by ubiquitination and degradation of StBAM1. Therefore, our study reveals the regulatory mechanism of SbRFP1 in the process of CIS and provides more powerful evidence for the effect of starch degradation on potato CIS.

Dynamics of the RING domain from human TRAF6 by 15N NMR spectroscopy: implications for biological function.

Activation of transcription factor NF-kappaB requires Lys63-linked polyubiquitination of the E3 ubiquitin ligase TRAF6 via protein-protein interactions mediated by a RING domain. In this study, intra- and intermolecular chemical exchange processes of the TRAF6 RING domain were analyzed by (15)N NMR spectroscopy. Micro- to millisecond time scale motions were assessed through R 1, R 2, NOE, and cross-correlated relaxation measurements, and the kinetics of these motions were quantified with relaxation dispersion. The relaxation experiments indicate that the protein core is rigid, consistent with the functional requirement that RING domains form a binding scaffold for E2 ubiquitin conjugation enzymes. Chemical exchange is observed at the C-terminal end of the main alpha-helix of the RING domain. The C-terminal end of the main alpha-helix from the RING domain is involved in E2-E3 interactions, and modulation of slow motions for this region of the helix may be a general mechanism by which these interactions achieve ubiquitin transfer. Chemical shift mapping indicates that the TRAF6 RING domain does not self-associate in solution. Numerous RING domains are homo- or heterodimeric, and this is thought to be a functional necessity for recruitment of substrates for ubiquitination, or recruitment of multiple E2 enzymes for efficient substrate ubiquitination. However, lack of self-association for the RING domain from TRAF6, and the observation that the intact protein is a trimer, suggests that close association of RING domains within a homodimeric scaffold may not be a fundamental requirement for biological function.

Functional analysis of a RING domain ankyrin repeat protein that is highly expressed during flower senescence.

A gene encoding a RING zinc finger ankyrin repeat protein (MjXB3), a putative E3 ubiquitin ligase, is highly expressed in petals of senescing four o'clock (Mirabilis jalapa) flowers, increasing >40,000-fold during the onset of visible senescence. The gene has homologues in many other species, and the Petunia homologue is strongly up-regulated in senescing Petunia corollas. Silencing the expression of this gene in Petunia, using virus-induced gene silencing, resulted in a 2 d extension in flower life. In Mirabilis, a 2 kb promoter region, 5' upstream of the MjXB3 gene, was isolated. The promoter sequence included putative binding sites for many DNA-binding proteins, including the bZIP, Myb, homeodomain-leucine zipper (HD-Zip), MADS-box, and WRKY transcription factors. The construct containing a 1 kb promoter region immediately upstream of the MjXB3 gene drove the strongest expression of the beta-glucuronidase (GUS) reporter gene in a transient expression assay. In Petunia, GUS expression under the control of this heterologous promoter fragment was specific to senescing flowers. The Mirabilis promoter GUS construct was tested in other flower species; while GUS activity in carnation petals was high during senescence, no expression was detected in three monocotyledonous flowers--daylily (Hemerocallis 'Stella d'Oro'), daffodil (Narcissus pseudonarcissus 'King Alfred'), and orchid (Dendrobium 'Emma White').

The activity of TRAF RING homo- and heterodimers is regulated by zinc finger 1.

Ubiquitin chains linked through lysine63 (K63) play a critical role in inflammatory signalling. Following ligand engagement of immune receptors, the RING E3 ligase TRAF6 builds K63-linked chains together with the heterodimeric E2 enzyme Ubc13-Uev1A. Dimerisation of the TRAF6 RING domain is essential for the assembly of K63-linked ubiquitin chains. Here, we show that TRAF6 RING dimers form a catalytic complex where one RING interacts with a Ubc13~Ubiquitin conjugate, while the zinc finger 1 (ZF1) domain and linker-helix of the opposing monomer contact ubiquitin. The RING dimer interface is conserved across TRAFs and we also show that TRAF5-TRAF6 heterodimers form. Importantly, TRAF5 can provide ZF1, enabling ubiquitin transfer from a TRAF6-bound Ubc13 conjugate. Our study explains the dependence of activity on TRAF RING dimers, and suggests that both homo- and heterodimers mediated by TRAF RING domains have the capacity to synthesise ubiquitin chains.

The PP4R1 subunit of protein phosphatase PP4 targets TRAF2 and TRAF6 to mediate inhibition of NF-κB activation.

TRAFs constitute a family of proteins that have been implicated in signal transduction by immunomodulatory cellular receptors and viral proteins. TRAF2 and TRAF6 have an E3-ubiquitin ligase activity, which is dependent on the integrity of their RING finger domain and it has been associated with their ability to activate the NF-κB and AP1 signaling pathways. A yeast two-hybrid screen with TRAF2 as bait, identified the regulatory subunit PP4R1 of protein phosphatase PP4 as a TRAF2-interacting protein. The interaction of TRAF2 with PP4R1 depended on the integrity of the RING finger domain of TRAF2. PP4R1 could interact also with the TRAF2-related factor TRAF6 in a RING domain-dependent manner. Exogenous expression of PP4R1 inhibited NF-κB activation by TRAF2, TRAF6, TNF and the Epstein-Barr virus oncoprotein LMP1. In addition, expression of PP4R1 downregulated IL8 induction by LMP1, whereas downregulation of PP4R1 by RNA interference enhanced the induction of IL8 by LMP1 and TNF. PP4R1 could mediate the dephosphorylation of TRAF2 Ser11, which has been previously implicated in TRAF2-mediated activation of NF-κB. Finally, PP4R1 could inhibit TRAF6 polyubiquitination, suggesting an interference with the E3 ubiquitin ligase activity of TRAF6. Taken together, our data identify a novel mechanism of NF-κB pathway inhibition which is mediated by PP4R1-dependent targeting of specific TRAF molecules.

Efficient mRNA polyadenylation requires a ubiquitin-like domain, a zinc knuckle, and a RING finger domain, all contained in the Mpe1 protein.

Almost all eukaryotic mRNAs must be polyadenylated at their 3' ends to function in protein synthesis. This modification occurs via a large nuclear complex that recognizes signal sequences surrounding a poly(A) site on mRNA precursor, cleaves at that site, and adds a poly(A) tail. While the composition of this complex is known, the functions of some subunits remain unclear. One of these is a multidomain protein called Mpe1 in the yeast Saccharomyces cerevisiae and RBBP6 in metazoans. The three conserved domains of Mpe1 are a ubiquitin-like (UBL) domain, a zinc knuckle, and a RING finger domain characteristic of some ubiquitin ligases. We show that mRNA 3'-end processing requires all three domains of Mpe1 and that more than one region of Mpe1 is involved in contact with the cleavage/polyadenylation factor in which Mpe1 resides. Surprisingly, both the zinc knuckle and the RING finger are needed for RNA-binding activity. Consistent with a role for Mpe1 in ubiquitination, mutation of Mpe1 decreases the association of ubiquitin with Pap1, the poly(A) polymerase, and suppressors of mpe1 mutants are linked to ubiquitin ligases. Furthermore, an inhibitor of ubiquitin-mediated interactions blocks cleavage, demonstrating for the first time a direct role for ubiquitination in mRNA 3'-end processing.

Ectopic expression of the RING domain of the Arabidopsis peroxin2 protein partially suppresses the phenotype of the photomorphogenic mutant de-etiolated1.

The Arabidopsis constitutive photomorphogenic/de-etiolated 1/FUSCA (COP/DET1/FUS) proteins repress photomorphogenesis by degrading positive regulators of photomorphogenesis, such as the transcription factor long hypocotyl5 (HY5). The gain-of-function mutant ted3, which partially suppresses the det1 mutant, contains a missense mutation of a Val-to-Met substitution before the C-terminal RING finger domain of the peroxisomal membrane protein peroxin2 (PEX2). We hypothesized that a truncated PEX2 protein, which only contains the C-terminal RING domain, is initiated by the ted3 mutation and by-passes the function of DET1 in the nucleus. Although we have not been able to detect this hypothetic peptide in vivo, we show in this study that, when fused with a fluorescent protein and overexpressed, the PEX2 RING domain can localize to the nucleus, where it is able to interact with HY5, and PEX2 RING domain overexpression in det1 also partially suppresses the det1 phenotype. Compared with det1, ted3 det1 plants have significantly decreased levels of the HY5 protein and the expression of most of the analyzed HY5 target genes is altered to levels comparable to those in hy5. We conclude that compromised activity of HY5 may have been mainly responsible for the partial reversal of the det1 phenotype in ted3 det1. Our data support the notion that, when appropriately localized, some RING finger domains may be able to achieve neomorphic effects in the cell.

Keeping cell identity in Arabidopsis requires PRC1 RING-finger homologs that catalyze H2A monoubiquitination.

Polycomb group (PcG) proteins form conserved regulatory complexes that modify chromatin to repress the genes that are not required in a specific differentiation status [1]. In animals, the two best-characterized PcG complexes are PRC2 and PRC1, which respectively possess histone 3 lysine 27 (H3K27) trimethyltransferase [2-4] and histone 2A lysine 119 (H2AK119) E3 ubiquitin ligase activities [5-7]. In Arabidopsis, PRC2 activity is also required for the gene silencing mechanism [8]; however, the existence of PRC1 has been questioned, because plant genomes do not encode clear PRC1 components and H2A monoubiquitination has not been detected [6, 9]. Conversely, recent reports have unveiled the presence of homologs to PRC1 components that together with plant-specific proteins could be part of the long-sought PRC1-like complexes [10, 11]. Here we show that the PRC1 RING-finger homologs AtBMI1A and AtBMI1B are implicated in the repression of embryonic and stem cell regulators. Plants impaired in AtBMI1A and AtBMI1B show derepression of embryonic traits in somatic cells, displaying a phenotype similar to plants mutant in PRC2 components [12-14]. Our data demonstrate that the AtBMI1A/B proteins mediate H2A monoubiquitination in Arabidopsis and that this mark, together with PRC2-mediated H3K27 trimethylation, plays a key role in maintaining cell identity.

Ring finger ubiquitin protein ligases and their implication to the pathogenesis of human diseases.

The ubiquitin proteasome system (UPS) plays a fundamental role in maintaining the correct balance of protein levels inside all living cells. Degradation of proteins by this pathway is essential for most cellular processes including cell signalling, DNA repair, apoptosis and gene transcription. Any disruption to the system is likely to have severe consequences which may lead to disorders including neurodegeneration and cancer. Ubiquitin protein ligases are a group of UPS proteins of particular importance because these proteins determine targeting specificity via recognition of a 'target' protein and its' subsequent 'tagging' with ubiquitin. The 26S proteasome recognises these mutli-ubiquitylated proteins, allowing the correct protein to be degraded at the correct time and place within each cell. Several types of ubiquitin protein ligase have now been identified, however, the largest group by far are those proteins containing a 'RING' motif. In this review, examples will be given whereby abnormal protein ubiquitylation due to absence or inefficiency of a RING protein ligase is proposed to be a key regulator of the disease process. Ways in which we may be able to reverse these effects or manipulate these proteins to restore function will be discussed.