A rice heterochronic mutant, mori1, is defective in the juvenile-adult phase change.

We have identified five recessive allelic mutations, mori1-1 to mori1-5, which drastically modify the shoot architecture of rice. The most remarkable feature of mori1 plants is a rapid production of small leaves and short branches. The mori1 plants are about 5 cm in height even 7 months after sowing. No reproductive growth was attained in mori1 plants even if inductive short-day treatment was applied. Leaves of mori1 at any position were very small and the size and shape were comparable to those of the wild-type 2nd leaf. The stem of mori1 7 months after sowing did not differentiate node and internode and had randomly oriented vascular bundles, which were characteristic of the basal part of the wild-type stem where 2nd and 3rd leaves were inserted. These structural characteristics indicate that mori1 maintains the 2nd-leaf stage (juvenile phase) of the wild type. The short plastochron and high cell division activity in the shoot apical meristem further confirmed the juvenility of mori1, corresponding to the 2nd-leaf-differentiation stage in the wild-type embryo. Furthermore, the apparent photosynthetic rate in mori1 leaves was low as in the wild-type 2nd leaf. Thus, mori1 is a heterochronic mutation that suppresses the induction of adult phase and the termination of the juvenile phase. Therefore, MORI1 plays an important role in the juvenile-adult phase change. The importance of heterochronic mutations in modifying shoot architecture is discussed.     

The rodent Four-jointed ortholog Fjx1 regulates dendrite extension

The extrinsic and intrinsic factors that regulate the size and complexity of dendritic arborizations are still poorly understood. Here we identify Fjx1, the rodent ortholog of the Drososphila planar cell polarity (PCP) protein Four-jointed (Fj), as a new inhibitory factor that regulates dendrite extension. The Drosophila gene four-jointed (fj) has been suggested to provide directional information in wing discs, but the mechanism how it acts is only poorly understood and the function of its mammalian homolog Fjx1 remains to be investigated. We analyzed the phenotype of a null mutation for mouse Fjx1. Homozygous Fjx1 mutants show an abnormal morphology of dendritic arbors in the hippocampus. In cultured hippocampal neurons from Fjx1 mutant mice, loss of Fjx1 resulted in an increase in dendrite extension and branching. Addition of Fjx1 to cultures of dissociated hippocampal neurons had the opposite effect and reduced the length of dendrites and decreased dendritic branching. Rescue experiments with cultured neurons showed that Fjx1 can act both cell-autonomously and non-autonomously. Our results identify Fjx1 as a new inhibitory factor that regulates dendrite extension.     

ARF1 regulates pH-dependent COP functions in the early endocytic pathway

Coat proteins of the COP family were recently shown by us and others to be involved in membrane transport in the endocytic pathway, in addition to their known functions in the biosynthetic pathway. We have also shown that membrane association of endosomal COPs depends on the acidic endosomal pH, in contrast to biosynthetic COPs. In this paper, we report that both membrane recruitment of endosomal COPs and in vitro biogenesis of transport intermediates destined for late endosomes, depend on a cytosolic factor, which we identified as the small GTP-binding protein ARF1. Our data indicate that ARF1 does not act via activation of an endosomal phospholipase D. We also find that ARF1 membrane association is regulated by the endosomal pH, and that this controls the pH-dependent association of endosomal COPs. These studies thus show that ARF1 regulates COP functions in the endocytic pathway, and indicate that ARF1 acts as the cytosolic component of a transmembrane pH-sensing mechanism.     

Phytochrome-mediated control of COP1 gene expression in rice plants

We isolated a COP1 cDNA from rice and found that it could complement the Arabidopsis cop1-4 mutant. The putative rice COP1 protein has the Ring-finger, coiled-coil. and WD-40 repeat domains, which are also conserved in pea, tomato, and mammalian COP1 proteins. The degree of overall identity between rice COP1 and Arabidopsis COP1 is 73%, and the similarity value is 83%. Expression of rice COP1 was detected in almost all plant tissues, with the level being relatively higher in calli and very low in etiolated leaves. The expression level was positively controlled by light in etiolated and green leaves. At the end of the light period, expression of the gene in green leaves could be down-regulated by far-red light. This far-red light effect could be prevented by subsequent irradiation with red light. These results indicate that phytochrome regulates rice COP1 expression.     

Myeloid leukemia factor 1 regulates p53 by suppressing COP1 via COP9 signalosome subunit 3

Myeloid leukemia factor 1 (MLF1) was first identified as the leukemic fusion protein NPM-MLF1 generated by the t(3;5)(q25.1;q34) chromosomal translocation. Although MLF1 expresses normally in a variety of tissues including hematopoietic stem cells and the overexpression of MLF1 correlates with malignant transformation in human cancer, little is known about how MLF1 is involved in the regulation of cell growth. Here we show that MLF1 is a negative regulator of cell cycle progression functioning upstream of the tumor suppressor p53. MLF1 induces p53-dependent cell cycle arrest in murine embryonic fibroblasts. This action requires a novel binding partner, subunit 3 of the COP9 signalosome (CSN3). A reduction in the level of CSN3 protein with small interfering RNA abrogated MLF1-induced G1 arrest and impaired the activation of p53 by genotoxic stress. Furthermore, ectopic MLF1 expression and CSN3 knockdown inversely affect the endogenous level of COP1, a ubiquitin ligase for p53. Exogenous expression of COP1 overcomes MLF1-induced growth arrest. These results indicate that MLF1 is a critical regulator of p53 and suggest its involvement in leukemogenesis through a novel CSN3-COP1 pathway.     

Hepatopoietin interacts directly with COP9 signalosome and regulates AP-1 activity

Hepatopoietin (HPO)/augmenter of liver regeneration (ALR) is a specific hepatotrophic growth factor, which plays a key role in liver regeneration. Our previous study indicated that HPO executes its function by an inter-reactive network of the autocrine, paracrine and endocrine pathways. Recently, we have demonstrated that intracellular HPO interacts with Jun activation domain-binding protein 1 (JAB1) and leads to potentiation of activating protein-1 (AP-1) activity in a MAPK independent fashion. JAB1 is the fifth subunit of the COP9 signalosome (CSN), which is first identified as a suppressor of plant morphogenesis. A protein complex kinase activity associated with the CSN has been reported but not identified yet. In this report, we investigated further the association of HPO with the whole CSN. HPO exists in a complex with the eight-component CSN, both when purified from glycerol gradient centrifugation and when reciprocal immunoprecipitated from the lysates of transfected COS-7 cells. Intracellular HPO colocalizes with endogenous CSN in nucleus of hepatic cells. In addition, intracellular function of HPO that increases the phosphorylation of c-Jun leading to potentiate the AP-1 activity is inhibited by curcumin, a potent inhibitor of CSN-associated kinase. Taken together, these results elucidate a novel relationship of intracellular growth factor, HPO with large protein complex, CSN, which suggests a possible linkage between CSN and liver regeneration.     

HASTY,the Arabidopsis ortholog of exportin 5/MSN5, regulates phase change and morphogenesis

Loss-of-function mutations of HASTY (HST) affect many different processes in Arabidopsis development. In addition to reducing the size of both roots and lateral organs of the shoot, hst mutations affect the size of the shoot apical meristem, accelerate vegetative phase change, delay floral induction under short days, adaxialize leaves and carpels, disrupt the phyllotaxis of the inflorescence, and reduce fertility. Double mutant analysis suggests that HST acts in parallel to SQUINT in the regulation of phase change and in parallel to KANADI in the regulation of leaf polarity. Positional cloning demonstrated that HST is the Arabidopsis ortholog of the importin beta-like nucleocytoplasmic transport receptors exportin 5 in mammals and MSN5 in yeast. Consistent with a potential role in nucleocytoplasmic transport, we found that HST interacts with RAN1 in a yeast two-hybrid assay and that a HST-GUS fusion protein is located at the periphery of the nucleus. HST is one of at least 17 members of the importin-beta family in Arabidopsis and is the first member of this family shown to have an essential function in plants. The hst loss-of-function phenotype suggests that this protein regulates the nucleocytoplasmic transport of molecules involved in several different morphogenetic pathways, as well as molecules generally required for root and shoot growth.     

The RING-Finger E3 Ubiquitin Ligase COP1 SUPPRESSOR1 Negatively Regulates COP1 Abundance in Maintaining COP1 Homeostasis in Dark-Grown Arabidopsis Seedlings

CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) functions as an E3 ubiquitin ligase in both plants and animals. In dark-grown Arabidopsis thaliana seedlings, COP1 targets photomorphogenesis-promoting factors for degradation to repress photomorphogenesis. Little is known, however, about how COP1 itself is regulated. Here, we identify COP1 SUPPRESSOR1 (CSU1), a RING-finger E3 ubiquitin ligase, as a regulator of COP1. Genetic evidence demonstrates that csu1 mutations suppress cop1-6 phenotypes completely in the dark. Furthermore, CSU1 colocalizes with COP1 in nuclear speckles and negatively regulates COP1 protein accumulation in darkness. CSU1 can ubiquitinate COP1 in vitro and is essential for COP1 ubiquitination in vivo. Therefore, we conclude that CSU1 plays a major role in maintaining COP1 homeostasis by targeting COP1 for ubiquitination and degradation in dark-grown seedlings.     

The COP9 signalosome regulates cell proliferation of Dictyostelium discoideum

Regulated protein destruction involving SCF (Skp1/Cullin/F-box, E3 ubiquitin ligase) complexes is required for multicellular development of Dictyostelium discoideum. Dynamic modification of cullin by nedd8 is required for the proper action of SCF. The COP9 signalosome (CSN), first identified in a signaling pathway for light response in plants, functions as a large multi-protein complex that regulates cullin neddylation in eukaryotes. Still, there is extreme sequence divergence of CSN subunits of the yeasts in comparison to the multicellular plants and animals. Using the yeast two-hybrid system, we have identified the CSN5 subunit as a potential interacting partner of a cell surface receptor of Dictyostelium. We further identified and characterized all 8 CSN subunits in Dictyostelium discoideum. Remarkably, despite the ancient origin of Dictyostelium, its CSN proteins cluster very closely with their plant and animal counterparts. We additionally show that the Dictyostelium subunits, like those of other systems are capable of multi-protein interactions within the CSN complex. Our data also indicate that CSN5 (and CSN2) are essential for cell proliferation in Dictyostelium, a phenotype similar to that of multicellular organisms, but distinct from that of the yeasts. Finally, we speculate on a potential role of CSN in cullin function and regulated protein destruction during multicellular development of Dictyostelium.     

The C. elegans glycopeptide hormone receptor ortholog, FSHR-1, regulates germline differentiation and survival

BACKGROUND: The mammalian glycopeptide hormone receptors (GPHRs) are key regulators of reproductive development, and their homologs are widely distributed throughout the animal kingdom. The C. elegans genome encodes a single GPHR family member, FSHR-1, which shares equal identity to the FSH, LH, and TSH receptors from mammals. RESULTS: Because loss of fshr-1 function does not produce a visible phenotype in C. elegans, we conducted a genome-wide RNAi-feeding screen to identify genes that perform functions that overlap with those of fshr-1. This approach led to the identification of the PUF family members fbf-1 and fbf-2 (the fbfs). Whereas a weak reduction in fbf activity caused little or no discernable effect in the wild-type, an equivalent loss in the fshr-1(0) mutant background resulted in a highly penetrant germline-masculinization phenotype. Furthermore, many fshr-1(0);fbf(RNAi) animals failed to maintain a germline stem cell niche. We also show that fshr-1 and the fbfs promote germline survival and prevent apoptosis with fog-1 and fog-3 and that simultaneous loss of fshr-1 and the fbfs can override the canonical requirement for fog-1 and fog-3 in the execution of the male-germline fate. Finally, we provide evidence that FSHR-1 controls germline processes nonautonomously via the soma and that FSHR-1 acts through a canonical signaling pathway involving Galpha(s) and adenyl cyclase. CONCLUSIONS: Our results indicate a conserved role for GPHR family receptors in controlling germline development and fertility. Our data suggest a model whereby FSHR-1 signaling acts in parallel to the known sex-determination pathway to control multiple aspects of germline development.     

Blue light-dependent interaction of CRY2 with SPA1 regulates COP1 activity and floral initiation in Arabidopsis

Cryptochromes are blue light receptors that mediate light regulation of gene expression in all major evolution lineages, but the molecular mechanism underlying cryptochrome signal transduction remains not fully understood. It has been reported that cryptochromes suppress activity of the multifunctional E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) to regulate gene expression in response to blue light. But how plant cryptochromes mediate light suppression of COP1 activity remains unclear. We report here that Arabidopsis CRY2 (cryptochrome 2) undergoes blue light-dependent interaction with the COP1-interacting protein SUPPRESSOR OF PHYTOCHROME A 1 (SPA1). We demonstrate that SPA1 acts genetically downstream from CRY2 to mediate blue light suppression of the COP1-dependent proteolysis of the flowering-time regulator CONSTANS (CO). We further show that blue light-dependent CRY2-SPA1 interaction stimulates CRY2-COP1 interaction. These results reveal for the first time a wavelength-specific mechanism by which a cryptochrome photoreceptor mediates light regulation of protein degradation to modulate developmental timing in Arabidopsis.     

The C. elegans MELK ortholog PIG-1 regulates cell size asymmetry and daughter cell fate in asymmetric neuroblast divisions

In the nematode Caenorhabditis elegans, neurons are generated from asymmetric divisions in which a mother cell divides to produce daughters that differ in fate. Here, we demonstrate that the gene pig-1 regulates the asymmetric divisions of neuroblasts that divide to produce an apoptotic cell and either a neural precursor or a neuron. In pig-1 mutants, these neuroblasts divide to produce daughters that are more equal in size, and their apoptotic daughters are transformed into their sisters, leading to the production of extra neurons. PIG-1 is orthologous to MELK, a conserved member of the polarity-regulating PAR-1/Kin1/SAD-1 family of serine/threonine kinases. Although MELK has been implicated in regulating the cell cycle, our data suggest that PIG-1, like other PAR-1 family members, regulates cell polarity.     

Role of rice PPS in late vegetative and reproductive growth

The rice peter pan syndrome-1 (pps-1) mutant shows a prolonged juvenile phase and early flowering. Although the early vegetative phase and flowering time of pps-1 have been closely examined, the phenotypes in the late vegetative and reproductive phases are not yet well understood. In the ninth leaf blade of pps-1, the relative length of the midrib was comparable to the sixth leaf blade of wild-type. Moreover, pps-1 had a small inflorescence meristem and small panicles. These phenotypes indicate that in pps-1 the juvenile phase coexists with the late vegetative phase, resulting in small panicles. Gibberellin is known to promote the juvenile-adult phase transition. d18-k is dwarf and has a prolonged juvenile phase. Double mutant (d18-k pps-1) showed the same phenotype as the pps-1, indicating that PPS is upstream of GA biosynthetic genes.     

The COP9 signalosome regulates Skp2 levels and proliferation of human cells

The COP9 signalosome (CSN) is a conserved, multisubunit complex first identified as a developmental regulator in plants. Gene inactivation of single CSN subunits results in early embryonic lethality in mice, indicating that the CSN is essential for mammalian development. The pleiotropic function of the CSN may be related to its ability to remove the ubiquitin-like peptide Nedd8 from cullin-RING ubiquitin ligases, such as the SCF complex, and therefore regulate their activity. However, the mechanism of CSN regulatory action on cullins has been debated, since, paradoxically, the CSN has an inhibitory role in vitro, while genetic evidence supports a positive regulatory role in vivo. We have targeted expression of CSN subunits 4 and 5 in human cells by lentivirus-mediated small hairpin RNA delivery. Down-regulation of either subunit resulted in disruption of the CSN complex and in Cullin1 hyperneddylation. Functional consequences of CSN down-regulation were decreased protein levels of Skp2, the substrate recognition subunit of SCF(Skp2), and stabilization of a Skp2 target, the cyclin-dependent kinase inhibitor p27(Kip1). CSN down-regulation caused an impairment in cell proliferation, which could be partially reversed by suppression of p27(Kip1). Moreover, restoring Skp2 levels in CSN-deficient cells recovered cell cycle progression, indicating that loss of Skp2 in these cells plays an important role in their proliferation defect. Our data indicate that the CSN is necessary to ensure the assembly of a functional SCF(Skp2) complex and therefore contributes to cell cycle regulation of human cells.     

Fanconi anemia ortholog FANCM regulates meiotic crossover distribution in plants

Meiotic recombination increases genetic diversity and manipulation of its frequency and distribution holds great promise in crop breeding. In Arabidopsis thaliana, FANCM (a homolog of mammalian Fanconi anemia complementation group M) suppresses recombination and its function seems conserved in other species including the rosids Brassica spp. and pea (Pisum sativum), and the monocot rice (Oryza sativa). To examine the role of FANCM during meiotic recombination in lettuce (Lactuca sativa, an asterid), we characterized the function of lettuce LsFANCM and found that it can functionally substitute for AtFANCM in transgenic Arabidopsis plants. Moreover, three independent CRISPR/Cas9-edited lettuce Lsfancm mutants showed reduced pollen viability and seed setting. Unexpectedly, analyses of chromosome behavior revealed that 77.8% of Lsfancm meiocytes exhibited univalents. The normal formation of double-strand breaks in DNA and the discontinuous assembly of synaptonemal complex in Lsfancm mutants supports the hypothesis that LsFANCM might be dispensable for the initiation of meiotic recombination but required for normal synapsis. Furthermore, the frequency of lettuce HEI10 (Human Enhancer of Invasion 10) foci, a marker for Class-I crossovers (COs), was similar between wild-type (WT) and Lsfancm. Strikingly, the distribution of LsHEI10 foci and chiasmata in Lsfancm meiotic chromosomes was markedly different from the WT. A similar alteration in the distribution of Class-I COs was also observed in the Arabidopsis Atfancm mutant. Taken together, these results demonstrate that FANCM is important for shaping the distribution of meiotic Class-I COs in plants, and reveal an evolutionarily divergent role for FANCM in meiotic bivalent formation between Arabidopsis and lettuce.

FANCM has a role in shaping meiotic crossover in plants, and its function in meiotic bivalent formation has diverged in Arabidopsis and lettuce.     

The Drosophila ortholog of the endolysosomal membrane protein, endolyn, regulates cell proliferation

Endolyn (CD164) is a sialomucin that regulates the proliferation, adhesion, and migration of human haematopoietic stem and progenitor cells. This molecule is predominately localized in endocytotic compartments, where it may contribute to endolysosomal biogenesis and trafficking. In order to more closely define the function of endolyn from an evolutionary view-point, we first analyzed endolyn orthologs in species ranging from insects, fish, and birds to mammals. The predicted molecular structures of the endolyn orthologs from these species are well conserved, particularly with respect to significant O-linked glycosylation of the extracellular domain, and the high degree of amino acid similarities within their transmembrane and cytoplasmic domains, with the latter possessing the lysosomal target signal, YXXphi. Focusing on Drosophila, our studies showed that the subcellular distribution of endolyn in non-polarized Drosophila S2 cells resembles that of its human counterpart in hematopoietic cells, with its predominant localization being within intracellular vesicles, while a small fraction occurs on the cell surface. Both Y --> A and L --> A mutations in the YHTL motif perturbed the normal subcellular distribution of Drosophila endolyn. Interestingly, embryonic and early larval development was often arrested in endolyn-deficient Drosophila mutants. This may partly be due to the role of endolyn in regulating cell proliferation, since knock-down of endolyn expression in S2 cells resulted in up to 50% inhibition of cell growth, with a proportion of cells undergoing apoptosis. Taken together, these results demonstrate that endolyn is an evolutionarily conserved sialomucin fundamentally involved in cell proliferation in both the human and Drosophila melanogaster.     

The COP9 signalosome regulates cell proliferation of Dictyostelium discoideum

Regulated protein destruction involving SCF (Skp1/Cullin/F-box, E3 ubiquitin ligase) complexes is required for multicellular development of Dictyostelium discoideum. Dynamic modification of cullin by nedd8 is required for the proper action of SCF. The COP9 signalosome (CSN), first identified in a signaling pathway for light response in plants, functions as a large multi-protein complex that regulates cullin neddylation in eukaryotes. Still, there is extreme sequence divergence of CSN subunits of the yeasts in comparison to the multicellular plants and animals. Using the yeast two-hybrid system, we have identified the CSN5 subunit as a potential interacting partner of a cell surface receptor of Dictyostelium. We further identified and characterized all 8 CSN subunits in Dictyostelium discoideum. Remarkably, despite the ancient origin of Dictyostelium, its CSN proteins cluster very closely with their plant and animal counterparts. We additionally show that the Dictyostelium subunits, like those of other systems are capable of multi-protein interactions within the CSN complex. Our data also indicate that CSN5 (and CSN2) are essential for cell proliferation in Dictyostelium, a phenotype similar to that of multicellular organisms, but distinct from that of the yeasts. Finally, we speculate on a potential role of CSN in cullin function and regulated protein destruction during multicellular development of Dictyostelium.