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In nature, plants are exposed to several environmental stresses that can be continuous or recurring. Continuous stress can be lethal, but stress after priming can increase the tolerance of a plant to better prepare for future stresses. Reports have suggested that transcription factors are involved in stress memory after recurrent stress; however, less is known about the factors that regulate the resetting of stress memory. Here, we uncovered a role for Constitutive Photomorphogenesis 5A (CSN5A) in the regulation of stress memory for resetting transcriptional memory genes (APX2 and HSP22) and H3K4me3 following recurrent heat stress. Furthermore, CSN5A is also required for the deposition of H3K4me3 following recurrent heat stress. Thus, CSN5A plays an important role in the regulation of histone methylation and transcriptional stress memory after recurrent heat stress.

Motivation The COP9 signalosome is a highly conserved multi-protein complex consisting of eight subunits, which influences key developmental pathways through its regulation of protein stability and transcription. In Arabidopsis thaliana, mutations in the COP9 signalosome exhibit a number of diverse pleiotropic phenotypes. Total or partial loss of COP9 signalosome function in Arabidopsis leads to misregulation of a number of genes involved in DNA methylation, suggesting that part of the pleiotropic phenotype is due to global effects on DNA methylation. Results We determined and analyzed the methylomes and transcriptomes of both partial- and total-loss-of-function Arabidopsis mutants of the COP9 signalosome. Our results support the hypothesis that the COP9 signalosome has a global genome-wide effect on methylation and that this effect is at least partially encoded in the DNA. Our analyses suggest that COP9 signalosome-dependent methylation is related to gene expression regulation in various ways. Differentially methylated regions tend to be closer in the 3D conformation of the genome to differentially expressed genes. These results suggest that the COP9 signalosome has a more comprehensive effect on gene expression than thought before, and this is partially related to regulation of methylation. The high level of COP9 signalosome conservation among eukaryotes may also suggest that COP9 signalosome regulates methylation not only in plants but also in other eukaryotes, including humans. Supplementary information Supplementary data are available at Bioinformatics online.

The COP9 signalosome protein complex has a central role in the regulation of development of multicellular organisms. While the function of this complex in ubiquitin-mediated protein degradation is well established, results over the past few years have hinted that the COP9 signalosome may function more broadly in the regulation of gene expression. Here, using DamID technology, we show that COP9 signalosome subunit 7 functionally associates with a large number of genomic loci in the Drosophila genome, and show that the expression of many genes within these loci is COP9 signalosome-dependent. This association is likely direct as we show CSN7 binds DNA in vitro. The genes targeted by CSN7 are preferentially enriched for transcriptionally active regions of the genome, and are involved in the regulation of distinct gene ontology groupings including imaginal disc development and cell-cycle control. In accord, loss of CSN7 function leads to cell-cycle delay and altered wing development. These results indicate that CSN7, and by extension the entire COP9 signalosome, functions directly in transcriptional control. While the COP9 signalosome protein complex has long been known to regulate protein degradation, here we expand the role of this complex by showing that subunit 7 binds DNA in vitro and functions directly in vivo in transcriptional control of developmentally important pathways that are relevant for human health.

The COP9 (constitutive photomorphogenesis 9) signalosome (CSN) is an evolutionarily conserved protein complex which regulates various growth and developmental processes. However, the role of CSN during environmental stress is largely unknown. Using Arabidopsis as model organism, we used CSN hypomorphic mutants to study the role of the CSN in plant responses to environmental stress and found that heat stress specifically enhanced the growth of csn5a-1 but not the growth of other hypomorphic photomorphogenesis mutants tested. Following heat stress, csn5a-1 exhibits an increase in cell size, ploidy, photosynthetic activity, and number of lateral roots and an upregulation of genes connected to the auxin response. Immunoblot analysis revealed an increase in deneddylation of CUL1 but not CUL3 following heat stress in csn5a-1, implicating improved CUL1 activity as a basis for the improved growth of csn5a-1 following heat stress. Studies using DR5::N7-VENUS and DII-VENUS reporter constructs confirm that the heat-induced growth is due to an increase in auxin signaling. Our results indicate that CSN5A has a specific role in deneddylation of CUL1 and that CSN5A is required for the recovery of AUX/IAA repressor levels following recurrent heat stress to regulate auxin homeostasis in Arabidopsis.

The ubiquitin–proteasome system plays a major role in the rhythmic accumulation and turnover of molecular clock components. In turn, these ∼24 h molecular rhythms drive circadian rhythms of behavior and physiology. InDrosophila, the ubiquitin–proteasome system also plays a critical role in light-dependent degradation of the clock protein Timeless (TIM), a key step in the entrainment of the molecular clocks to light–dark cycles. Here, we investigated the role of the COP9 signalosome (CSN), a general regulator of protein degradation, in fly circadian rhythms. We found that null mutations in the genes encoding the CSN4 and CSN5 subunits prevent normal TIM degradation by light in the pacemaker lateral neurons (LNs) as does LN-specific expression of a dominant-negativeCSN5transgene. These defects are accompanied by strong reductions in behavioral phase shifts of adult flies lacking normal CSN5 activity in LNs. Defects in TIM degradation and resetting of behavioral phases were rescued by overexpression of Jetlag (JET), the F-box protein required for light-mediated TIM degradation. Flies lacking normal CSN activity in all clock neurons are rhythmic in constant light, a phenotype previously associated withjetmutants. Together, these data indicate that JET and the CSN lie in a common pathway leading to light-dependent TIM degradation. Surprisingly, we found that manipulations that strongly inhibit CSN activity had minimal effects on circadian rhythms in constant darkness, indicating a specific role for the CSN in light-mediated TIM degradation.

The COP9 Signalosome (CSN) is a multiprotein complex that was originally identified in Arabidopsis thaliana as a negative regulator of photomorphogenesis and subsequently shown to be a general eukaryotic regulator of developmental signaling. The CSN plays various roles, but it has been most often implicated in regulating protein degradation pathways. Six of eight CSN subunits bear a sequence motif called PCI. Here, we report studies of subunit 7 (CSN7) from Arabidopsis, which contains such a motif. Our in vitro and structural results, based on 1.5 Å crystallographic data, enable a definition of a PCI domain, built from helical bundle and winged helix subdomains. Using functional binding assays, we demonstrate that the PCI domain (residues 1 to 169) interacts with two other PCI proteins, CSN8 and CSN1. CSN7 interactions with CSN8 use both PCI subdomains. Furthermore, we show that a C-terminal tail outside of this PCI domain is responsible for association with the non-PCI subunit, CSN6. In vivo studies of transgenic plants revealed that the overexpressed CSN7 PCI domain does not assemble into the CSN, nor can it complement a null mutation of CSN7. However, a CSN7 clone that contains the PCI domain plus part of the CSN6 binding domain can complement the null mutation in terms of seedling viability and photomorphogenesis. These transgenic plants, though, are defective in adult growth, suggesting that the CSN7 C-terminal tail plays additional functional roles. Together, the findings have implications for CSN assembly and function, highlighting necessary interactions between subunits.

The COP9 signalosome (CSN) is a multisubunit regulator highly conserved in evolution. We show here that CSN subunit 8 (CSN8) is essential for Drosophila development. CSN8 is maternally contributed and present throughout development. Null mutants generated in this study are larval lethal, showing phenotypes associated with mutations in either CSN4 (molting defects) or CSN5 (melanotic tumors). Analysis of mitotic and germ-line csn8(null) clones revealed the requirement of CSN8 for multiple developmental processes. The germ-line clones arrested at mid-oogenesis, while the mitotic clones led to deformed adult eyes or wings. CSN8 is present exclusively as part of the CSN holo-complex, and lack of CSN8 in the mutants leads to CSN instability. Consistent with this, Cullin deneddylation is impaired in the csn8(null) mutants.

The roles of individual eukaryotic translation initiation factor 3 (eIF3) subunits are largely unclear, although some are essential, while others are thought to have regulatory roles. The 'e' subunit, also known as Int-6/Int6, is a candidate for a regulatory subunit as it is not essential for translation initiation in yeasts. eIF3e associates with the COP9 signalosome, and localizes to the nucleus in certain tissues. To further elucidate the roles of eIF3e, we have taken a genetic approach using Arabidopsis as a model system. Overexpression of eIF3e results in defects similar to mutations in the COP9 signalosome. eIF3e protein, but not transcript, over accumulates in csn mutants, and eIF3e is degraded in a proteasome-dependent fashion. In vitro and in vivo assays suggest that excess eIF3e inhibits translation. We conclude that the COP9 signalosome maintains a precise regulation of eIF3e levels, which is necessary for normal development in Arabidopsis.

The COP9 signalosome is a multifunctional regulator essential for Drosophila development. A loss-of-function mutant in Drosophila COP9 signalosome subunit 5 (CSN5) develops melanotic bodies, a phenotype common to mutants in immune signaling. csn5(null) larvae accumulated high levels of Cactus that co-localizes with Dorsal to the nucleus. However, Dorsal-dependent transcriptional activity remained repressed in the absence of an inducing signal, despite its nuclear localization. Dorsal activity in mutant larvae and NFkappaB activity in CSN5 down-regulated mammalian cells can be induced following activation of the Toll/IL-1 pathway. csn5(null) larvae contained more hemocytes than wild-type (wt) larvae. A large portion of these cells have differentiated to lamellocytes (LM), a hemocyte cell type rarely seen in normal larvae. The results presented here indicate that CSN5 is a negative regulator of Dorsal subcellular localization, and of hemocyte proliferation and differentiation. These results further indicate that nuclear localization of Dorsal can be uncoupled from its activation. Surprisingly, CSN5 is not necessary for immune-induced degradation of Cactus.

The DNA damage response is vigorously activated by DNA double-strand breaks (DSBs). The chief mobilizer of the DSB response is the ATM protein kinase. We discovered that the COP9 signalosome (CSN) is a crucial player in the DSB response and an ATM target. CSN is a protein complex that regulates the activity of cullin ring ubiquitin ligase (CRL) complexes by removing the ubiquitin-like protein, NEDD8, from their cullin scaffold. We find that the CSN is physically recruited to DSB sites in a neddylation-dependent manner, and is required for timely repair of DSBs, affecting the balance between the two major DSB repair pathways—nonhomologous end-joining and homologous recombination repair (HRR). The CSN is essential for the processivity of deep end-resection—the initial step in HRR. Cullin 4a (CUL4A) is recruited to DSB sites in a CSN- and neddylation-dependent manner, suggesting that CSN partners with CRL4 in this pathway. Furthermore, we found that ATM-mediated phosphorylation of CSN subunit 3 on S410 is critical for proper DSB repair, and that loss of this phosphorylation site alone is sufficient to cause a DDR deficiency phenotype in the mouse. This novel branch of the DSB response thus significantly affects genome stability. España Ministerio de Economía y Competitividad SAF2013-43255-P

The COP9 signalosome (CSN) is a multisubunit protein complex that intersects the ubiquitin (Ub)-proteasome pathway at several junctions. The CSN associates with several protein kinases that target key regulatory proteins that themselves are targets for Ub-dependent degradation, and several subunits themselves are phosphoproteins. We previously reported that Arabidopsis CSN7 is a phosphoprotein. To identify the kinases responsible for CSN7 phosphorylation, we biochemically purified CSN7-kinsae activities from cauliflower through a four-step purification procedure that resulted in a ca. 300-fold enrichment. One of these activities is Ca 2+ dependent, while the second is not. Peptide fingerprint analysis of the purified proteins identified three putative CSN7 kinases.

TRC8 encodes an E3-ubiquitin ligase disrupted in a family with hereditary renal cell carcinoma (RCC). We previously reported that Drosophila Trc8 (DTrc8) overexpression inhibits growth and that human and fly proteins interact with with the COP9 signalosome (CSN) subunit JAB1/CSN5. However, further mechanistic evidence linking DTrc8 growth suppression to CSN5 was lacking. Here, we show that haploinsufficiency of CSN5, or a T100I point mutation (CSN5(3)), relieved growth suppression by DTrc8, whereas CSN5(1) (E160V) and CSN5(2) (G147D) mutations had no effect. The strength of yeast two-hybrid interactions between DTrc8 and CSN5 were in complete agreement with the observed phenotypes. DTrc8 overexpression resulted in elevated levels of CSN5 and CSN7, but had no effect on NEDD8-modified Cul-1. In contrast to CSN5, heterozygosity for CSN4null had no effect on the DTrc8 phenotype. We also looked for genetic interactions between DTrc8 and other MPN domain proteins in the CSN and 26S proteasome lid. CSN6 haploinsufficiency restored growth, whereas reduction of proteasome subunits RPN8 or RPN11 had no effect. DTrc8 expression increased the level of digitonin-extractable CSN complex, consistent with elevated levels of CSN5 and 7. Our genetic results confirm that DTrc8-induced growth suppression is CSN5 (and CSN6) dependent. While there was no obvious influence on CSN deneddylation activity, the increase in CSN subunits and holocomplex suggests that TRC8 modulates signalosome levels or compartmentalization.

The COP9 Signalosome (CSN), a highly conserved eight-subunit complex, is found in all higher eukaryotes. It contains eight core subunits, named CSN1-8, in order of decreasing molecular weight. The CSN is structurally similar to the regulatory lid of 26S proteasome and the eukaryotic translation initiation factor eIF3. CSN is also now known to play an essential role in signaling processes controlling many aspects of plant and Drosophila development. Taken together, the various genetic studies demonstrate that the CSN is involved at the nexus between multiple signal inputs and a variety of downstream regulatory cascades controlling specific aspects of cellular differentiation. Research in various organisms has converged onto the notion that CSN is biochemically linked to ubiquitin-dependent protein degradation. Other proposed roles for the CSN include regulating eIF3 and kinase signaling. CSN is itself is both a target for kinase activity and associates with and coordinates activity of kinases. CSN-associated kinases. This kinase activity further regulates the ubiquitin-dependent degradation of various transcription factors. This review concentrates on the proposed activity of the CSN as a regulator of protein phosphorylation.

The COP9 signalosome (CSN) is an essential eight-subunit repressor of light-regulated development in Arabidopsis. This complex has also been identified in animals, though its developmental role remains obscure. CSN subunits have been implicated in various cellular processes, suggesting a possible role for the CSN as an integrator of multiple signaling pathways. In order to elucidate the function of the CSN in animals, a Drosophila model system has previously been established. Gel-filtration analysis with antibodies against CSN subunits 4, 5 and 7 revealed that these proteins act as a complex in Drosophila that is similar in size to the plant and mammalian complexes. Null mutations in either one of two subunits, CSN4 or CSN5, are larval lethal. Successful embryogenesis appears to be a consequence of maternal contribution of the complex. Biochemical analysis indicates that the different subunits are found in both CSN-dependent and CSN-independent forms, and that these forms are differentially affected by the mutations. Phenotypic characterization of these two mutants indicates that they show both shared and unique phenotypes, which suggest specific roles for each subunit. Both mutants have defective oocyte and embryo patterning, and defects in response to DNA damage, while csn5 mutants develop melanotic tumors and csn4 mutants have phenotypes reminiscent of defects in ecdysone signaling.

The expression of light-regulated genes in plants is controlled by different classes of photoreceptors that act through a variety of signaling molecules. During photomorphogenesis, the early light-induced protein (Elip) genes are among the first to be induced. To understand the light signal transduction pathways that regulate Elip expression, the twoElip genes, Elip1 andElip2, in Arabidopsis were studied, taking advantage of the genetic tools available for studying light signaling in Arabidopsis. Using two independent quantitative reverse transcriptase-PCR techniques, we found that red, far-red, and blue lights positively regulate expression of the Elip genes. Phytochrome A and phytochrome B are involved in this signaling. The cryptochrome or phototropin photoreceptors are not required for blue-light induction of either Elip gene, suggesting the involvement of an additional, unidentified, blue-light receptor. Although the COP9 signalosome, a downstream regulator, is involved in dark repression of both Elips, Elip1 andElip2 show different expression patterns in the dark. The transcription factor HY5 promotes the light induction ofElip1, but not Elip2. A defect in photosystem II activity in greening of hy5 seedlings may result from the loss of Elip1. Heat shock positively controlled Elip1 and Elip2 in a light-independent fashion. This induction is independent of HY5, indicating that heat shock and light activate transcription of the Elip genes through independent pathways.

Three protein complexes (the proteasome regulatory lid, the COP9 signalosome and eukaryotic translation initiation factor 3) contain protein subunits with a well defined protein domain, the PCI domain. At least two (the COP9 signalosome and the lid) appear to share a common evolutionary origin. Recent advances in our understanding of the structure and function of the three complexes point to intriguing and unanticipated connections between the cellular functions performed by these three protein assemblies, especially between translation initiation and proteolytic protein degradation.

The COP9 signalosome has eight core subunits that are highly conserved between plants and animals. Some of the subunits in Arabidopsis are found in forms that are independent of the complex. The COP9 complex is essential for animal development. The COP9 signalosome may have both an evolutionary and a physical relationship with both the regulatory lid of the proteasome and eIF3.

Similarities between the three related ‘PCI' complexes – eIF3, the COP9 signalosome and the proteasome lid – have hinted at novel pathways controlling protein homeostasis. Recent experiments with fission yeast have begun to weigh in with genetic evidence.

The COP9 signalosome (CSN) is an evolutionarily conserved multi-protein complex that interfaces with the ubiquitin-proteasome pathway and plays critical developmental roles in both animals and plants. Although some subunits are present only in an ∼320-kDa complex-dependent form, other subunits are also detected in configurations distinct from the 8-subunit holocomplex. To date, the only known biochemical activity intrinsic to the complex, deneddylation of the Cullin subunits from Cullin-RING ubiquitin ligases, is assigned to CSN5. As an essential step to understanding the structure and assembly of a CSN5-containing subcomplex of the CSN, we reconstituted a CSN4-5-6-7 subcomplex. The core of the subcomplex is based on a stable heterotrimeric association of CSN7, CSN4, and CSN6, requiring coexpression in a bacterial reconstitution system. To this heterotrimer, we could then add CSN5 in vitro to reconstitute a quaternary complex. Using biochemical and biophysical methods, we identified pairwise and combinatorial interactions necessary for the formation of the CSN4-5-6-7 subcomplex. The subcomplex is stabilized by three types of interactions: MPN-MPN between CSN5 and CSN6, PCI-PCI between CSN4 and CSN7, and interactions mediated through the CSN6 C terminus with CSN4 and CSN7. CSN8 was also found to interact with the CSN4-6-7 core. These data provide a strong framework for further investigation of the organization and assembly of this pivotal regulatory complex.

The COP9 Signalosome protein complex (CSN) is a pleiotropic regulator of plant development and contains eight-subunits. Six of these subunits contain the PCI motif which mediates specific protein interactions necessary for the integrity of the complex. COP9 complex subunit 7 (CSN7) contains an N-terminal PCI motif followed by a C-terminal extension which is also necessary for CSN function. A yeast-interaction trap assay identified the small subunit of ribonucelotide reductase (RNR2) from Arabidopsis as interacting with the C-terminal section of CSN7. This interaction was confirmed in planta by both bimolecular fluorescence complementation and immuoprecipitation assays with endogenous proteins. The subcellular localization of RNR2 was primarily nuclear in meristematic regions, and cytoplasmic in adult cells. RNR2 was constitutively nuclear in csn7 mutant seedlings, and was also primarily nuclear in wild type seedlings following exposure to UV-C. These two results correlate with constitutive expression of several DNA-damage response genes in csn7 mutants, and to increased tolerance of csn7 seedlings to UV-C treatment. We propose that the CSN is a negative regulator of RNR activity in Arabidopsis.

The COP9 signalosome (CSN) is a highly conserved protein complex that was originally described as a repressor of light-dependent growth and transcription in Arabidopsis. The most studied CSN function is the regulation of protein degradation, which occurs primarily through the removal of the ubiquitin-like modifier Nedd8 from cullin-based E3 ubiquitin ligases. This activity can regulate transcription-factor stability and, therefore, transcriptional activity. Recent data suggest that the CSN also regulates transcription on the chromatin by mechanisms that are not yet clearly understood. Furthermore, the CSN subunits CSN5 and CSN2 seem to act as transcriptional coactivators and corepressors, respectively. Here, I re-evaluate the mechanisms by which the CSN acts as a transcriptional regulator, and suggest that they could extend beyond the regulation of protein stability.

The roles of individual Eukaryotic translation Initiation Factor 3 (eIF3) subunits are largely unclear. Though some are essential, while others are thought to have regulatory roles. The “e” subunit, also known as Int-6, is a candidate for a regulatory subunit as it is not essential for translation initiation in yeasts. To further elucidate the roles of eIF3e, we have employed an interaction-trap screen using the yeast two-hybrid system. eIF3e interacts in yeast with subunits of the ribosome, COP9 signalosome and 26S proteasome. These interactions mesh well with our recent results which showed that eIF3e is degraded in a CSN-dependent, proteasome-dependent fashion, and inhibits translation when present in excess.

The COP9 signalosome (CSN), an eight-subunit protein complex, is conserved in all higher eukaryotes. CSN intersects the ubiquitin–proteasome pathway, modulating signaling pathways controlling various aspects of development. We are using Drosophila as a model system to elucidate the function of this important complex. Transcriptome data were generated for four csn mutants, sampled at three developmental time points. Our results are highly reproducible, being confirmed using two different experimental setups that entail different microarrays and different controls. Our results indicate that the CSN acts as a transcriptional repressor during development of Drosophila, resulting in achronic gene expression in the csn mutants. ‘Time shift' analysis with the publicly available Drosophila transcriptome data indicates that genes repressed by the CSN are normally induced primarily during late embryogenesis or during metamorphosis. These temporal shifts are likely due to the roles of the CSN in regulating transcription factors. A null mutation in CSN subunit 4 and hypomorphic mutations in csn5 lead to more severe defects than seen in the csn5-null mutants strain, suggesting that CSN5 carries only some of the CSN function.

A core fragment of Arabidopsis thaliana COP9 signalosome (CSN) subunit 7 was expressed in Escherichia coli. The protein was purified to homogeneity and screened for crystallization. Crystallization conditions were refined using the sitting-drop vapour-diffusion method. Crystals were obtained using polyethylene glycol 8000 as a precipitant and have a thick rod-like morphology. Their crystallographic symmetry is orthorhombic, space group C222(1), with unit-cell parameters a = 57.2, b = 86.2, c = 72.6 A and a diffraction limit of 2.06 A.

We study gene expression data, derived from developing tissues, under multiple genetic backgrounds (mutations). Motivated by the perceived behavior under these background, our main goals are to explore time windows questions: Find a large set of genes that have a similar behavior in two different genetic backgrounds, under an appropriate time shift. Find a model that approximates the dynamics of a gene network in developing tissues at different continuous time windows. We first explain the biological significance of these problems, and then explore their computational complexity, which ranges from polynomial to NP-hard. We developed algorithms and heuristics for the different problems, and ran those on synthetic and biological data, with very encouraging results.

The COP9 signalosome (CSN) was identified close to a decade ago ([Wei et al., 1994][1]). The biochemical purification of the CSN ([Chamovitz et al., 1996][2]) was a major breakthrough, because it showed that Arabidopsis genetics could be approached biochemically, and it subsequently lead to the

Different classes of photoreceptor neurons (R cells) in the Drosophila compound eye form connections in different optic ganglia. The R1-R6 subclass connects to the first optic ganglion, the lamina, and relies upon glial cells as intermediate targets. Conversely, R cells promote glial cell development including migration of glial cells into the target region. Here, we show that the JAB1/CSN5 subunit of the COP9 signalosome complex is expressed in R cells, accumulates in the developing optic lobe neuropil, and through the analysis of a unique set of missense mutations, is required in R cells to induce lamina glial cell migration. In these CSN5 alleles, R1-R6 targeting is disrupted. Genetic analysis of protein null alleles further revealed that the COP9 signalosome is required at an earlier stage of development for R cell differentiation.

The Jun activating binding protein (JAB1) specifically stabilizes complexes of c-Jun or JunD with AP-1 sites, increasing the specificity of target gene activation by AP-1 proteins. JAB1 is also known as COP9 signalosome subunit 5 (CSN5), which is a component of the COP9 signalosome regulatory complex (CSN). Over the past year, JAB1/CSN5 has been implicated in numerous signaling pathways including those that regulate light signaling in plants, larval development in Drosophila, and integrin signaling, cell cycle control, and steroid hormone signaling in a number of systems. However, the general role of the CSN complex, and the specific role of JAB1/CSN5, still remain obscure. This review attempts to integrate the available data to help explain the role of JAB1/CSN5 and the COP9 signalosome in regulating multiple pathways (in this review, both JAB1 and CSN5 terminologies are used interchangeably, depending on the source material).

The Arabidopsis COP9 signalosome is a multisubunit repressor of photomorphogenesis that is conserved among eukaryotes. This complex may have a general role in development. As a step in dissecting the biochemical mode of action of the COP9 signalosome, we determined the sequence of proteins that copurify with this complex. Here we describe the association between components of the COP9 signalosome (CSN1, CSN7, and CSN8) and two subunits of eukaryotic translation initiation factor 3 (eIF3), eIF3e (p48, known also as INT-6) and eIF3c (p105). To obtain a biochemical marker for Arabidopsis eIF3, we cloned the Arabidopsis ortholog of the eIF3 subunit eIF3b (PRT1). eIF3e coimmunoprecipitated with CSN7, and eIF3c coimmunoprecipitated with eIF3e, eIF3b, CSN8, and CSN1. eIF3e directly interacted with CSN7 and eIF3c. However, eIF3e and eIF3b cofractionated by gel filtration chromatography in a complex that was larger than the COP9 signalosome. Whereas eIF3, as detected through eIF3b, localized solely to the cytoplasm, eIF3e, like CSN7, was also found in the nucleus. This suggests that eIF3e and eIF3c are probably components of multiple complexes and that eIF3e and eIF3c associate with subunits of the COP9 signalosome, even though they are not components of the COP9 signalosome core complex. This interaction may allow for translational control by the COP9 signalosome.

The CONSTITUTIVE PHOTOMORPHOGENIC9 (COP9) complex is a nuclear localized, multisubunit protein complex essential for repression of light-mediated development in Arabidopsis. Mutations that abolish the complex result in constitutive photomorphogenic development in darkness and pleiotropic developmental defects in both light and darkness. Here, we report the identification of two apparently redundant genes, AJH1 and AJH2, that encode a subunit of the COP9 complex. Both AJH1 and AJH2 share high amino acid sequence identity (62 and 63%, respectively) with JAB1, a specific mammalian coactivator of AP-1 transcription. The proteins encoded by these two genes are present in both complex and monomeric forms, whereas complex formation is in part mediated by the direct interaction with FUSCA6. In addition, the stability of the monomeric AJH proteins requires functional COP1 and DEETIOLATED1 loci. Together with the fact that the previously known subunit FUSCA6 is an Arabidopsis homolog of human GPS1, a negative regulator of AP-1 transcription, our data suggest that the COP9 complex may contain both negative and positive regulators of transcription. Therefore, the COP9 complex may achieve its pleiotropic effects on Arabidopsis development by modulating activities of transcription factors in response to environmental stimuli.

Environmental light signals are sensed by multiple families of photoreceptors and transduced by largely unknown mechanisms to regulate plant development. In this report, genetic analysis suggested that light signals perceived by both phytochromes and a blue light receptor converge to repress the action of Arabidopsis COP9 in suppressing seedling photomorphogenesis. Molecular cloning of the gene revealed that COP9 encodes a novel protein of 197 amino acids whose expression is not regulated by light. COP9 functions as a large (>560 kDa) complex(es) that is probably subjected to light modulation. In addition, COP8 and COP11 are required for either the COP9 complex formation or its stability. Therefore COP9, together with COP8 and COP11, defines a novel signaling step in mediating light control of plant development.

Int6 is a proto-oncogene implicated in various types of cancer, but the mechanisms underlying its activity are not clear. Int6 encodes a subunit of the eukaryotic translation initiation factor 3, and interacts with two related complexes, the proteasome, whose activity is regulated by Int6 in S. pombe, and the COP9 signalosome. The COP9 signalosome regulates the activity of Cullin-Ring Ubiquitin Ligases via deneddylation of their cullin subunit. We report here the generation and analysis of two Drosophila mutants in Int6. The mutants are lethal demonstrating that Int6 is an essential gene. The mutant larvae accumulate high levels of non-neddylated Cul1, suggesting that Int6 is a positive regulator of cullin neddylation. Overexpression in Int6 in cell culture leads to accumulation of neddylated cullins, further supporting a positive role for Int6 in regulating neddylation. Thus Int6 and the COP9 signalosome play opposing roles in regulation of cullin neddylation.

The COP9 signalosome (originally described as the COP9 complex) is an essential multi-subunit repressor of light-regulated development in plants [1] [2]. It has also been identified in mammals, though its role remains obscure [3] [4] [5]. This complex is similar to the regulatory lid of the proteasome and eIF3 [5] [9] [10] [11] [12] and several of its subunits are known to be involved in kinase signaling pathways [4] [6] [7] [8]. No proteins homologous to COP9 signalosome components were identified in the Saccharomyces cerevisiae genome, suggesting that the COP9 signalosome is specific for multi-cellular differentiation [13]. In order to reveal the developmental function of the COP9 signalosome in animals, we have isolated Drosophila melanogaster genes encoding eight subunits of the COP9 signalosome, and have shown by co-immunoprecipitation and gel-filtration analysis that these proteins are components of the Drosophila COP9 signalosome. Yeast two-hybrid assays indicated that several of these proteins interact, some through the PCI domain. Disruption of one of the subunits by either a P-element insertion or deletion of the gene caused lethality at the late larval or pupal stages. This lethality is probably a result of numerous pleiotropic effects. Our results indicate that the COP9 signalosome is conserved in invertebrates and that it has an essential role in animal development.

Arabidopsis COP9 is a component of a large protein complex that is essential for the light control of a developmental switch and whose conformation or size is modulated by light. The complex is acidic, binds heparin, and is localized within the nucleus. Biochemical purification of the complex to near homogeneity revealed that it contains 12 distinct subunits. One of the other subunits is COP11, mutations in which result in a phenotype identical to cop9 mutants. The COP9 complex may act to regulate the nuclear abundance of COP1, an established repressor of photomorphogenic development. During the biogenesis of the COP9 complex, a certain degree of prior subunit association is a prerequisite for proper nuclear translocation. Since both COP9 and COP11 have closely related human counterparts, the COP9 complex probably represents a conserved developmental regulator in higher eukaryotes.

TheCSN and the lid subcomplex ofthe proteasome share remarkablestructural and architecturalsimilarities, an identical number ofsubunits, common structuralmotifs, similar subunitinteractions, and a predominantlocalization to the nuclearperiphery. Interaction of lidsubunit Rpn6 with CSN1 suggestscross-talk between the twocomplexes.