Search

SUMMARYThe oncogenic GPCR Smoothened is a key transducer of the Hedgehog morphogen, which plays essential roles in the patterning of epithelial structures. Here, we examine how Hedgehog controls Smoothened subcellular localization and activity in a polarized epithelium using the Drosophila wing imaginal disc as a model. We provide evidence that Hedgehog promotes the stabilization of Smoothened by switching its fate after endocytosis toward recycling. This effect involves the sequential and additive action of Protein Kinase A, Casein Kinase I and the Fused kinase. Moreover, in the presence of very high levels of Hedgehog, a second effect of Fused leads to the local enrichment of Smoothened in the most basal domain of the cell membrane. Together, these results link the morphogenetic effects of Hedgehog to the apico-basal distribution of Smoothened and provide a novel mechanism for regulation of a GPCR by plasma membrane subcompartimentalisation.

Morphogen gradients need to be robust, but may also need to be tailored for specific tissues. Often this type of regulation is carried out by negative regulators and negative feedback loops. In the Hedgehog (Hh) pathway, activation of patched (ptc) in response to Hh is part of a negative feedback loop limiting the range of the Hh morphogen. Here, we show that in the Drosophila wing imaginal disc two other known Hh targets genes feed back to modulate Hh signaling. First, anterior expression of the transcriptional repressor Engrailed modifies the Hh gradient by attenuating the expression of the Hh pathway transcription factor cubitus interruptus (ci), leading to lower levels of ptc expression. Second, the E-3 ligase Roadkill shifts the competition between the full-length activator and truncated repressor forms of Ci by preferentially targeting full-length Ci for degradation. Finally, we provide evidence that Suppressor of fused, a negative regulator of Hh signaling, has an unexpected positive role, specifically protecting full-length Ci but not the Ci repressor from Roadkill.

Hedgehog (Hh) pathway inhibition by the conserved protein Suppressor of Fused (SuFu) is crucial to vertebrate development. By constrast, SuFu loss-of-function mutant has little effect in drosophila. Previous publications showed that the crystal structures of human and drosophila SuFu consist of two ordered domains that are capable of breathing motions upon ligand binding. However, the crystal structure of human SuFu does not give information about twenty N-terminal residues (IDR1) and an eighty-residue-long region predicted as disordered (IDR2) in the C-terminus, whose function is important for the pathway repression. These two intrinsically disordered regions (IDRs) are species-dependent. To obtain information about the IDR regions, we studied full-length SuFu's structure in solution, both with circular dichroism and small angle X-ray scattering, comparing drosophila, zebrafish and human species, to better understand this considerable difference. Our studies show that, in spite of similar crystal structures restricted to ordered domains, drosophila and vertebrate SuFu have very different structures in solution. The IDR2 of vertebrates spans a large area, thus enabling it to reach for partners and be accessible for post-translational modifications. Furthermore, we show that the IDR2 region is highly conserved within phyla but varies in length and sequence, with insects having a shorter disordered region while that of vertebrates is broad and mobile. This major variation may explain the different phenotypes observed upon SuFu removal.

Smaug is a conserved translational repressor that recognizes specific RNA motifs in a large number of mRNAs, including nuclear transcripts that encode mitochondrial enzymes. Smaug orthologs have been shown to form membraneless organelles (MLOs) in several organisms and cell types. Using single-molecule FISH we show here that SDHB and UQCRC1 mRNAs associate with Smaug1 MLOs in the human cell line U2OS. Simultaneous loss of function of Smaug1 and Smaug2 affects both mitochondrial respiration and mitochondrial network morphology. Deletion of specific Smaug1 protein regions resulted in impaired MLO formation that correlates with mitochondrial defects. In addition, rotenone but not the respiratory chain uncoupling agent CCCP rapidly induces Smaug1 MLO dissolution. Finally, metformin elicits a similar effect on Smaug1 MLOs and provokes the release of bounded mRNAs. We propose that mitochondrial activity affects Smaug1 MLO dynamics, thus allowing for regulation of nuclear mRNAs that encode key mitochondrial proteins. Fil: Fernández Alvarez, Ana Julia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Thomas, Maria Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Pascual, Malena Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Habif, Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina Fil: Pimentel, Jerónimo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Corbat, Agustín Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina Fil: Pessoa, Joao. Universidad de Lisboa; Portugal Fil: la Spina, Pablo Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Boscaglia, Lara. Fundación Instituto Leloir; Argentina Fil: Plessis, Anne. Sorbonne University; Francia. Université Paris Diderot - Paris 7; Francia Fil: Carmo Fonseca, Maria. Universidad de Lisboa; Portugal Fil: Grecco, Hernan Edgardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina Fil: Casado Pinna, Marta. Instituto de Biomedicina de Valencia; España Fil: Boccaccio, Graciela Lidia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina

From fly to mammals, the Smaug/Samd4 family of prion-like RNA-binding proteins control gene expression by destabilizing and/or repressing the translation of numerous target transcripts. However, the regulation of its activity remains poorly understood. We show that Smaug's protein levels and mRNA repressive activity are downregulated by Hedgehog signaling in tissue culture cells. These effects rely on the interaction of Smaug with the G-protein coupled receptor Smoothened, which promotes the phosphorylation of Smaug by recruiting the kinase Fused. The activation of Fused and its binding to Smaug are sufficient to suppress its ability to form cytosolic bodies and to antagonize its negative effects on endogenous targets. Importantly, we demonstrate in vivo that HH reduces the levels of smaug mRNA and increases the level of several mRNAs downregulated by Smaug. Finally, we show that Smaug acts as a positive regulator of Hedgehog signaling during wing morphogenesis. These data constitute the first evidence for a post-translational regulation of Smaug and reveal that the fate of several mRNAs bound to Smaug is modulated by a major signaling pathway.

Environmental gene regulatory influence networks (EGRINs) coordinate the timing and rate of gene expression in response to environmental signals. EGRINs encompass many layers of regulation, which culminate in changes in accumulated transcript levels. Here, we inferred EGRINs for the response of five tropical Asian rice (Oryza sativa) cultivars to high temperatures, water deficit, and agricultural field conditions by systematically integrating time-series transcriptome data, patterns of nucleosome-free chromatin, and the occurrence of known cis-regulatory elements. First, we identified 5447 putative target genes for 445 transcription factors (TFs) by connecting TFs with genes harboring known cis-regulatory motifs in nucleosome-free regions proximal to their transcriptional start sites. We then used network component analysis to estimate the regulatory activity for each TF based on the expression of its putative target genes. Finally, we inferred an EGRIN using the estimated transcription factor activity (TFA) as the regulator. The EGRINs include regulatory interactions between 4052 target genes regulated by 113 TFs. We resolved distinct regulatory roles for members of the heat shock factor family, including a putative regulatory connection between abiotic stress and the circadian clock. TFA estimation using network component analysis is an effective way of incorporating multiple genome-scale measurements into network inference.

African rice (Oryza glaberrima Steud.) is a cereal crop species closely related to Asian rice (Oryza sativa L.) but was independently domesticated in West Africa ∼3,000 years ago. African rice is rarely grown outside sub-Saharan Africa but is of global interest because of its tolerance to abiotic stresses. Here we describe a map of 2.32 million SNPs of African rice from whole-genome resequencing of 93 landraces. Population genomic analysis shows a population bottleneck in this species that began ∼13,000-15,000 years ago with effective population size reaching its minimum value ∼3,500 years ago, suggesting a protracted period of population size reduction likely commencing with predomestication management and/or cultivation. Genome-wide association studies (GWAS) for six salt tolerance traits identify 11 significant loci, 4 of which are within ∼300 kb of genomic regions that possess signatures of positive selection, suggesting adaptive geographical divergence for salt tolerance in this species.

The hot ABA-deficiency suppressor2 (has2) mutation increases drought tolerance and the ABA sensitivity of stomata closure and seed germination. Here we report that the HAS2 locus encodes the MITOCHONDRIAL EDITING FACTOR11 (MEF11), also known as LOVASTATIN INSENSITIVE1. has2/mef11 mutants exhibited phenotypes very similar to the ABA-hypersensitive mutant, hai1-1 pp2ca-1 hab1-1 abi1-2, which is impaired in four genes encoding type 2C protein phosphatases (PP2C) that act as upstream negative regulators of the ABA signaling cascade. Like pp2c, mef11 plants were more resistant to progressive water stress and seed germination was more sensitive to paclobutrazol (a gibberellin biosynthesis inhibitor) as well as mannitol and NaCl, compared with the wild-type plants. Phenotypic alterations in mef11 were associated with the lack of editing of transcripts for the mitochondrial cytochrome c maturation FN2 (ccmFN2) gene, which encodes a cytochrome c-heme lyase subunit involved in cytochrome c biogenesis. Although the abundance of electron transfer chain complexes was not affected, their dysfunction could be deduced from increased respiration and altered production of hydrogen peroxide and nitric oxide in mef11 seeds. As minor defects in mitochondrial respiration affect ABA signaling, this suggests an essential role for ABA in mitochondrial retrograde regulation.

Smoothened (SMO) is a G-protein-coupled receptor-related protein required for the transduction of Hedgehog (HH). The HH gradient leads to graded phosphorylation of SMO, mainly by the PKA and CKI kinases. How thresholds in HH morphogen regulate SMO to promote switch-like transcriptional responses is a central unsolved issue. Using the wing imaginal disc model in Drosophila, we identified new SMO phosphosites that enhance the effects of the PKA/CKI kinases on SMO accumulation, its localization at the plasma membrane and its activity. Surprisingly, phosphorylation at these sites is induced by the kinase Fused (FU), a known downstream effector of SMO. In turn, activation of SMO induces FU to act on its downstream targets. Overall, our data provide evidence for a SMO/FU positive regulatory loop nested within a multikinase phosphorylation cascade. We propose that this complex interplay amplifies signaling above a threshold that allows high HH signaling.

Smoothened (SMO) is a G-protein-coupled receptor-related protein required for the transduction of Hedgehog (HH). The HH gradient leads to graded phosphorylation of SMO, mainly by the PKA and CKI kinases. How thresholds in HH morphogen regulate SMO to promote switch-like transcriptional responses is a central unsolved issue. Using the wing imaginal disc model in

Environmental Gene Regulatory Influence Networks (EGRINs) coordinate the timing and rate of gene expression in response to environmental and developmental signals. EGRINs encompass many layers of regulation, which culminate in changes in the level of accumulated transcripts. Here we infer EGRINs for the response of five tropical Asian rice cultivars to high temperatures, water deficit, and agricultural field conditions, by systematically integrating time series transcriptome data (720 RNA-seq libraries), patterns of nucleosome-free chromatin (18 ATAC-seq libraries), and the occurrence of known cis-regulatory elements. First, we identify 5,447 putative target genes for 445 transcription factors (TFs) by connecting TFs with genes with known cis-regulatory motifs in nucleosome-free chromatin regions proximal to transcriptional start sites (TSS) of genes. We then use network component analysis to estimate the regulatory activity for these TFs from the expression of these putative target genes. Finally, we inferred an EGRIN using the estimated TFA as the regulator. The EGRIN included regulatory interactions between 4,052 target genes regulated by 113 TFs. We resolved distinct regulatory roles for members of a large TF family, including a putative regulatory connection between abiotic stress and the circadian clock, as well as specific regulatory functions for TFs in the drought response. TFA estimation using network component analysis is an effective way of incorporating multiple genome-scale measurements into network inference and that supplementing data from controlled experimental conditions with data from outdoor field conditions increases the resolution for EGRIN inference.

Plants rely on transcriptional dynamics to respond to multiple climatic fluctuations and contexts in nature. We analyzed the genome-wide gene expression patterns of rice (Oryza sativa) growing in rainfed and irrigated fields during two distinct tropical seasons and determined simple linear models that relate transcriptomic variation to climatic fluctuations. These models combine multiple environmental parameters to account for patterns of expression in the field of co-expressed gene clusters. We examined the similarities of our environmental models between tropical and temperate field conditions, using previously published data. We found that field type and macroclimate had broad impacts on transcriptional responses to environmental fluctuations, especially for genes involved in photosynthesis and development. Nevertheless, variation in solar radiation and temperature at the timescale of hours had reproducible effects across environmental contexts. These results provide a basis for broad-based predictive modeling of plant gene expression in the field. DOI: http://dx.doi.org/10.7554/eLife.08411.001, eLife digest Plants need to be able to sense and respond to changes in temperature, light levels and other aspects of their environment. One way in which plants can rapidly respond to these changes is to modify how genes involved in growth and other processes are expressed. Therefore, understanding how this happens may help us to improve the ability of crops to grow when exposed to drought or other extreme environmental conditions. Most previous studies into the effect of the environment on plant gene expression have been carried out under controlled conditions in a laboratory. These findings cannot reflect the full range of gene expression patterns that occur in the natural environment, where multiple factors (e.g. sunlight, water, nutrients) may vary at the same time. Therefore, it is important to also analyze the effect of fluctuations in multiple environmental factors in more complex field experiments. Plessis et al. developed mathematical models to analyze the gene expression patterns of rice plants grown in the tropical environment of the Philippines using two different farming practices. One field of rice was flooded and constantly supplied with fresh water (referred to as the irrigated field), while the other field was dry and only received water from rainfall (the rainfed field). The experiments show that temperature and levels of sunlight (including UV radiation) have a strong impact on gene expression in the rice plants. Short-term variations in temperature and sunlight levels also have the most consistent effect across the different fields and seasons tested. However, for many genes, the plants grown in the irrigated field responded to the changes in environmental conditions in a different way to the plants grown in the rainfed field. Further analysis identified groups of genes whose expression combined responses to several environmental factors at the same time. For example, certain genes that responded to increases in sunlight in the absence of drought responded to both sunlight levels and the shortage of water when a drought occurred. The next step is to test more types of environments and climates to be able to predict gene expression responses under future climatic conditions. DOI: http://dx.doi.org/10.7554/eLife.08411.002

International audience; Wheat (Triticum aestivum L.) grain storage proteins (GSPs) are major determinants of flour end-use value. Biological and molecular mechanisms underlying the developmental and nutritional determination of GSP accumulation in cereals are as yet poorly understood. Here we timed the accumulation of GSPs during wheat grain maturation relative to changes in metabolite and transcript pools in different conditions of nitrogen (N) and sulfur (S) availability. We found that the N/S supply ratio modulated the duration of accumulation of S-rich GSPs and the rate of accumulation of S-poor GSPs. These changes are likely to be the result of distinct relationships between N and S allocation, depending on the S content of the GSP. Most developmental and nutritional modifications in GSP synthesis correlated with the abundance of structural gene transcripts. Changes in the expression of transport and metabolism genes altered the concentrations of several free amino acids under variable conditions of N and S supply, and these amino acids seem to be essential in determining GSP expression. The comprehensive data set generated and analyzed here provides insights that will be useful in adapting fertilizer use to variable N and S supply, or for breeding new cultivars with balanced and robust GSP composition.

Defects in the biogenesis of or transport through primary cilia affect Hedgehog protein signaling, and many Hedgehog pathway components traffic through or accumulate in cilia. The Hedgehog receptor, Patched, negatively regulates the activity and ciliary accumulation of Smoothened, a seven transmembrane protein that is essential for transducing the Hedgehog signal. We found that this negative regulation of Smoothened required the ciliary localization of Patched, as specified either by its own cytoplasmic tail or by provision of heterologous ciliary localization signals. Surprisingly, given that Hedgehog binding promotes the exit of Patched from the cilium, we observed that an altered form of Patched that is retained in the cilium nevertheless responded to Hedgehog, resulting in Smoothened activation. Our results indicate that, whereas ciliary localization of Patched is essential for suppression of Smoothened activation, the primary event enabling Smoothened activation is binding of Hedgehog to Patched, and Patched ciliary removal is secondary.

Background: In Drosophila and vertebrates, suppressor of fused (Su(fu)) proteins act as negative regulators of the Gli/Ci transcription factors, which mediate the transcriptional effects of Hh signalling. Results: We sought for novel partners of Su(fu) in fly using the two-hybrid method. Most of the Su(fu) interactors thus identified are (or are likely to be) able to enter the nucleus. We focused on one of these putative partners, dMLF, which resembles vertebrate myelodysplasia/myeloid leukaemia factors 1 and 2. We demonstrate that dMLF binds specifically to Su(fu) in vitro and in vivo. Using a novel anti-dMLF antibody, we showed, that dMLF is a nuclear, chromosome-associated protein. We over-expressed a dMLF transgene in fly using an inducible expression system and showed that dMLF over-expression disrupts normal development, leading to either a lethal phenotype or adult structural defects associated with apoptosis and increased DNA synthesis. Furthermore, the dMLF-induced eye phenotype is enhanced by the loss of Su(fu) function, suggesting a genetic interaction between Su(fu) and dMLF. Conclusion: We propose that dSu(fu) and dMLF act together at the transcriptional level to coordinate patterning and proliferation during development.

International audience

Wheat grain storage protein (GSP) content and composition are the main determinants of the end-use value of bread wheat (Triticum aestivum L.) grain. The accumulation of glutenins and gliadins, the two main classes of GSP in wheat, is believed to be mainly controlled at the transcriptional level through a network of transcription factors. This regulation network could lead to stable cross-environment allometric scaling relationships between the quantity of GSP classes/subunits and the total quantity of nitrogen per grain. This work conducted a genetic mapping study of GSP content and composition and allometric scaling parameters of grain N allocation using a bread wheat worldwide core collection grown in three environments. The core collection was genotyped with 873 markers for genome-wide association and 167 single nucleotide polymorphism markers in 51 candidate genes for candidate association. The candidate genes included 35 transcription factors (TFs) expressed in grain. This work identified 74 loci associated with 38 variables, of which 19 were candidate genes or were tightly linked with candidate genes. Besides structural GSP genes, several loci putatively trans-regulating GSP accumulation were identified. Seven candidate TFs, including four wheat orthologues of barley TFs that control hordein gene expression, were associated or in strong linkage disequilibrium with markers associated with the composition or quantity of glutenin or gliadin, or allometric grain N allocation parameters, confirming the importance of the transcriptional control of GSP accumulation. Genome-wide association results suggest that the genes regulating glutenin and gliadin compositions are mostly distinct from each other and operate differently.

The genome of the human immunodeficiency virus type 1 (HIV-1) encodes the canonical retroviral proteins, as well as additional accessory proteins that enhance the expression of viral genes, the infectivity of the virus and the production of virions. The accessory Viral Protein U (Vpu), in particular, enhances viral particle production, while also promoting apoptosis of HIV-infected human T lymphocytes. Some Vpu effects rely on its interaction with the ubiquitin–proteasome protein degradation system, but the mechanisms responsible for its pro-apoptotic effects in vivo are complex and remain largely to be elucidated. We took advantage of the Drosophila model to study the effects of Vpu activity in vivo. Expression of Vpu in the developing Drosophila wing provoked tissue loss due to caspase-dependent apoptosis. Moreover, Vpu induced expression of the pro-apoptotic gene reaper, known to down-regulate Inhibitor of Apoptosis Proteins (IAPs) which are caspase-antagonizing E3 ubiquitin ligases. Indeed, Vpu also reduced accumulation of Drosophila IAP1 (DIAP1). Though our results demonstrate a physical interaction between Vpu and the proteasome-addressing SLIMB/β-TrCP protein, as in mammals, both SLIMB/βTrCP-dependent and -independent Vpu effects were observed in the Drosophila wing. Lastly, the pro-apoptotic effect of Vpu in this tissue was abrogated upon inactivation of the c-Jun N-terminal Kinase (JNK) pathway. Our results in the fly thus provide the first functional evidence linking Vpu pro-apoptotic effects to activation of the conserved JNK pathway.

On water deficit, abscisic acid (ABA) induces stomata closure to reduce water loss by transpiration. To identify Arabidopsis thaliana mutants which transpire less on drought, infrared thermal imaging of leaf temperature has been used to screen for suppressors of an ABA-deficient mutant (aba3-1) cold-leaf phenotype. Three novel mutants, called hot ABA-deficiency suppressor (has), have been identified with hot-leaf phenotypes in the absence of the aba3 mutation. The defective genes imparted no apparent modification to ABA production on water deficit, were inherited recessively and enhanced ABA responses indicating that the proteins encoded are negative regulators of ABA signalling. All three mutants showed ABA-hypersensitive stomata closure and inhibition of root elongation with little modification of growth and development in non-stressed conditions. The has2 mutant also exhibited increased germination inhibition by ABA, while ABA-inducible gene expression was not modified on dehydration, indicating the mutated gene affects early ABA-signalling responses that do not modify transcript levels. In contrast, weak ABA-hypersensitivity relative to mutant developmental phenotypes suggests that HAS3 regulates drought responses by both ABA-dependent and independent pathways. has1 mutant phenotypes were only apparent on stress or ABA treatments, and included reduced water loss on rapid dehydration. The HAS1 locus thus has the required characteristics for a targeted approach to improving resistance to water deficit. In contrast to has2, has1 exhibited only minor changes in susceptibility to Dickeya dadantii despite similar ABA-hypersensitivity, indicating that crosstalk between ABA responses to this pathogen and drought stress can occur through more than one point in the signalling pathway.

L'article original est publié par The American Society of Plant Biologists; CUP-SHAPED COTYLEDON2 (CUC2) and the interacting microRNA miR164 regulate leaf margin dissection. Here, we further investigate the evolution and the specific roles of the CUC1 to CUC3 genes during Arabidopsis thaliana leaf serration. We show that CUC2 is essential for dissecting the leaves of a wide range of lobed/serrated Arabidopsis lines. Inactivation of CUC3 leads to a partial suppression of the serrations, indicating a role for this gene in leaf shaping. Morphometric analysis of leaf development and genetic analysis provide evidence for different temporal contributions of CUC2 and CUC3. Chimeric constructs mixing CUC regulatory sequences with different coding sequences reveal both redundant and specific roles for the three CUC genes that could be traced back to changes in their expression pattern or protein activity. In particular, we show that CUC1 triggers the formation of leaflets when ectopically expressed instead of CUC2 in the developing leaves. These divergent fates of the CUC1 and CUC2 genes after their formation by the duplication of a common ancestor is consistent with the signature of positive selection detected on the ancestral branch to CUC1. Combining experimental observations with the retraced origin of the CUC genes in the Brassicales, we propose an evolutionary scenario for the CUC genes.

SummaryHedgehog (HH) is a major secreted morphogen involved in development, stem cell maintenance and oncogenesis [1, 2]. In Drosophila wing imaginal discs, HH produced in the posterior compartment diffuses into the anterior compartment to control target gene transcription via the transcription factor Cubitus interruptus (CI). The first steps in the reception and transduction of the HH signal are mediated by its receptor Patched (PTC) [3] and the seven-transmembrane-domain protein Smoothened (SMO) [4, 5]. PTC and HH control SMO by regulating its stability, trafficking, and phosphorylation (for review, see [6]). SMO interacts directly with the Ser-Thr protein kinase Fused (FU) and the kinesin-related protein Costal2 (COS2), which interact with each other and with CI in an intracellular Hedgehog transducing complex [7–9].We show here that HH induces FU targeting to the plasma membrane in a SMO-dependent fashion and that, reciprocally, FU controls SMO stability and phosphorylation. FU anchorage to the membrane is sufficient to make it a potent SMO-dependent, PTC-resistant activator of the pathway. These findings reveal a novel positive-feedback loop in HH transduction and are consistent with a model in which FU and SMO, by mutually enhancing each other's activities, sustain high levels of signaling and render the pathway robust to PTC level fluctuations.

International audience; In human, the myeloid leukemia factor 1 (hMLF1) has been shown to be involved in acute leukemia, and mlf related genes are present in many animals. Despite their extensive representation and their good conservation, very little is understood about their function. In Drosophila, dMLF physically interacts with both the transcription regulatory factor DREF and an antagonist of the Hedgehog pathway, Suppressor of Fused, whose over-expression in the fly suppresses the toxicity induced by polyglutamine. No connection between these data has, however, been established. Here, we show that dmlf is widely and dynamically expressed during fly development. We isolated and analyzed the first dmlf mutants: embryos lacking maternal dmlf product have a low viability with no specific defect, and dmlf(-)- adults display weak phenotypes. We monitored dMLF subcellular localization in the fly and cultured cells. We were able to show that, although generally nuclear, dMLF can also be cytoplasmic, depending on the developmental context. Furthermore, two differently spliced variants of dMLF display differential subcellular localization, allowing the identification of regions of dMLF potentially important for its localization. Finally, we demonstrate that dMLF can act developmentally and postdevelopmentally to suppress neurodegeneration and premature aging in a cerebellar ataxia model.

The Suppressor of fused (Su(fu)) protein is known to be a negative regulator of Hedgehog (Hh) signal transduction in Drosophila imaginal discs and embryonic development. It is antagonized by the kinase Fused (Fu) since Su(fu) null mutations fully suppress the lack of Fu kinase activity. In this study, we overexpressed the Su(fu) gene in imaginal discs and observed opposing effects depending on the position of the cells, namely a repression of Hh target genes in cells receiving Hh and their ectopic expression in cells not receiving Hh. These effects were all enhanced in a fu mutant context and were suppressed by cubitus interruptus (Ci) overexpression. We also show that the Su(fu) protein is poly-phosphorylated during embryonic development and these phosphorylation events are altered in fu mutants. This study thus reveals an unexpected role for Su(fu) as an activator of Hh target gene expression in absence of Hh signal. Both negative and positive roles of Su(fu) are antagonized by Fused. Based on these results, we propose a model in which Su(fu) protein levels and isoforms are crucial for the modulation of the different Ci states that control Hh target gene expression.

The Drosophila (fruit fly) model system has been instrumental in our current understanding of human biology, development, and diseases. Here, we used a high-throughput yeast two-hybrid (Y2H)-based technology to screen 102 bait proteins from Drosophila melanogaster, most of them orthologous to human cancer-related and/or signaling proteins, against high-complexity fly cDNA libraries. More than 2300 protein-protein interactions (PPI) were identified, of which 710 are of high confidence. The computation of a reliability score for each protein-protein interaction and the systematic identification of the interacting domain combined with a prediction of structural/functional motifs allow the elaboration of known complexes and the identification of new ones. The full data set can be visualized using a graphical Web interface, the PIMRider (http://pim.hybrigenics.com), and is also accessible in the PSI standard Molecular Interaction data format. Our fly Protein Interaction Map (PIM) is surprisingly different from the one recently proposed by Giot et al. with little overlap between the two data sets. Analysis of the differences in data sets and methods suggests alternative strategies to enhance the accuracy and comprehensiveness of the post-genomic generation of broad-scale protein interaction maps.

In Drosophila and vertebrates, suppressor of fused (Su(fu)) proteins act as negative regulators of the Gli/Ci transcription factors, which mediate the transcriptional effects of Hh signalling.We sought for novel partners of Su(fu) in fly using the two-hybrid method. Most of the Su(fu) interactors thus identified are (or are likely to be) able to enter the nucleus. We focused on one of these putative partners, dMLF, which resembles vertebrate myelodysplasia/myeloid leukaemia factors 1 and 2. We demonstrate that dMLF binds specifically to Su(fu) in vitro and in vivo. Using a novel anti-dMLF antibody, we showed, that dMLF is a nuclear, chromosome-associated protein. We over-expressed a dMLF transgene in fly using an inducible expression system and showed that dMLF over-expression disrupts normal development, leading to either a lethal phenotype or adult structural defects associated with apoptosis and increased DNA synthesis. Furthermore, the dMLF-induced eye phenotype is enhanced by the loss of Su(fu) function, suggesting a genetic interaction between Su(fu) and dMLF.We propose that dSu(fu) and dMLF act together at the transcriptional level to coordinate patterning and proliferation during development.

Background Hedgehog signaling proteins play important roles in development by controlling growth and patterning in various animals including Drosophila and mammals. Hedgehog signaling triggers changes in responsive cells through a novel transduction mechanism that ultimately controls the transcription of specific target genes via the activity of zinc finger transcription factors of the Cubitus interruptus /GLI family. In flies, key Hedgehog signal transduction components have been identified including the kinesin-related protein Costal2, the serinethreonine kinase Fused, and the PEST-containing protein Suppressor of Fused. These proteins control Cubitus interruptus cleavage, nucleo-cytoplasmic localization and activation. In fly embryos, Costal2, Fused, Suppressor of Fused and Cubitus interruptus are associated in at least one cytoplasmic complex, which interacts with the microtubules in a Hedgehog-dependent manner. Results Here we identified and mapped direct interactions between Cos2, Fu, and Ci using an in vitro affinity assay and the yeast two-hybrid system. Conclusions Our results provide new insights into the possible mechanism of the cytosolic steps of Hedgehog transduction.

0960-9822 doi: DOI: 10.1016/S0960-9822(98)70227-1; The Hedgehog (Hh) family of signalling proteins [1] mediate inductive interactions either directly or by controlling the transcription of other secreted proteins through the action of Gli transcription factors, such as Cubitus interruptus (Ci) [2]. In Drosophila, the transcription of Hh targets requires the activation of the protein kinase Fused (Fu) and the inactivation of both Suppressor of fused (Su(fu)) and Costal-2 (Cos-2) [3]. Fu is required for Hh signalling in the embryo and in the wing imaginal disc and acts also as an antitumorigen in ovaries [4]. All fu– phenotypes are suppressed by the loss of function of Su(fu) [5]. Fu, Cos-2 and Ci are coassociated in vivo in large complexes that are bound to microtubules in a Hh-dependent manner [6,7]. Here we investigate the role of Su(fu) in the intracellular part of the Hh signalling pathway. Using the yeast two-hybrid method and an in vitro binding assay, we show that Su(fu), Ci and Fu can interact directly to form a trimolecular complex, with Su(fu) binding to both its partners simultaneously. Su(fu) and Ci also coimmunoprecipitate from embryo extracts. We propose that, in the absence of Hh signalling, Su(fu) inhibits Ci by binding to it and that, upon reception of the Hh signal, Fu is activated and counteracts Su(fu), leading to the activation of Ci.

In yeast, the fate of linear DNA molecules upon transformation is determined by the existence of sequence homology between chromosomes and the ends of the transforming molecule. To understand the mechanism of integration of transforming DNA, we have studied the influence of DNA concentration on the frequency and type of transformants obtained, using either non-replicative or replicative plasmids. In both cases, increasing DNA concentration results in multiple tandem repeats integrated into the chromosome containing the homologous target sequence. When a diploid strain is transformed, multiple tandem repeats occur in only one of the two homologous chromosomes at a time. The frequency distribution of the different types of integrants observed indicates non-independent integration events likely to result from plasmid-plasmid interaction prior to chromosome integration. In addition, our results define the proper conditions for optimized gene targetting or gene rescue experiments.

International audience; The Hedgehog (HH) signaling pathway is crucial for the development of many organisms and its inappropriate activation is involved in numerous cancers. HH signal controls the traffic and activity of the seven-pass transmembrane protein Smoothened (SMO), leading to the transcriptional regulation of HH-responsive genes. In Drosophila, the intracellular transduction events following SMO activation depend on cytoplasmic multimeric complexes that include the Fused (FU) protein kinase. Here we show that the regulatory domain of FU physically interacts with the last 52 amino acids of SMO and that the two proteins colocalize in vivo to vesicles. The deletion of this region of SMO leads to a constitutive activation of SMO, promoting the ectopic transcription of HH target genes. This activation is partially dependent of FU activity. Thus, we identify a novel link between SMO and the cytoplasmic complex(es) and reveal a negative role of the SMO C-terminal region that interacts with FU. We propose that FU could act as a switch, activator in presence of HH signal or inhibitor in absence of HH.

The Hedgehog (Hh) signaling pathway initiates an evolutionarily conserved developmental program required for the proper patterning of many tissues. Costal2 (Cos2) is a requisite component of the Hh pathway, whose mechanistic role is not well understood. Cos2 was initially predicted, based on its primary sequence, to function as a microtubule-associated (MT) molecular motor. However, despite being identified over a decade ago, evidence showing that Cos2 function might require kinesin-like properties has for the most part been lacking. Thus the prevailing dogma in the field is that Cos2 functions solely as a scaffolding protein during Hh signal transduction. Here, we provide the first evidence that Cos2 motility is required for its biological function, and that this motility may be Hh regulated. We show that Cos2 motility requires an active motor domain, ATP and microtubules. Additionally, Cos2 recruits and transports other components of the Hh signaling pathway, including the transcription factor Cubitus interruptus (Ci), throughout the cell. Drosophila expressing cos2 mutations that encode proteins that lack motility are attenuated in their ability to regulate Ci activity and exhibit phenotypes consistent with attenuated Cos2 function. Combined, these results demonstrate that Cos2 motility plays an important role in its function, regulating the amounts and activity of Ci that ultimately interpret the level of Hh to which cells are exposed.

Agrobacterium rhizogenes induces root formation and inserts a fragment of its plasmid into the genome of infected plants. A part of the transferred region (TL-region) of the Ri plasmid of A. rhizogenes strain A4 was cloned in pBR322. Insertions of the Escherichia coli lacZ coding region into the hybrid plasmids were made in vivo using mini-Mu-duction. Two mini-Mus were used, one with the Mu A and B transposase genes (MudII1681) and the other without (MudII1734). Two inserts which result in E. coli lacZ expression where shown to be located in the T-DNA region. This indicates that portions of the T-DNA are capable of expression in bacteria. When these two hybrid plasmids were transformed into Agrobacterium only the one harboring MudII1734 insert gave transformants which correspond to homologous recombination. These results indicate that gene fusion and insertion directed mutagenesis can be simultaneously obtained with this mini-Mu and could be used to study Agrobacterium gene expression.

19 páginas, 8 figuras, Smaug is a conserved translational regulator that binds numerous mRNAs, including nuclear transcripts that encode mitochondrial enzymes. Smaug orthologs form cytosolic membrane-less organelles (MLOs) in several organisms and cell types. We have performed single-molecule fluorescence in situ hybridization (FISH) assays that revealed that SDHB and UQCRC1 mRNAs associate with Smaug1 bodies in U2OS cells. Loss of function of Smaug1 and Smaug2 (also known as SAMD4A and SAMD4B, respectively) affected both mitochondrial respiration and morphology of the mitochondrial network. Phenotype rescue by Smaug1 transfection depends on the presence of its RNA-binding domain. Moreover, we identified specific Smaug1 domains involved in MLO formation, and found that impaired Smaug1 MLO condensation correlates with mitochondrial defects. Mitochondrial complex I inhibition upon exposure to rotenone, but not strong mitochondrial uncoupling upon exposure to CCCP, rapidly induced the dissolution of Smaug1 MLOs. Metformin and rapamycin elicited similar effects, which were blocked by pharmacological inhibition of AMP-activated protein kinase (AMPK). Finally, we found that Smaug1 MLO dissolution weakens the interaction with target mRNAs, thus enabling their release. We propose that mitochondrial respiration and the AMPK-mTOR balance controls the condensation and dissolution of Smaug1 MLOs, thus regulating nuclear mRNAs that encode key mitochondrial proteins. This article has an associated First Person interview with the first authors of the paper., This work was supported by the following grants: PICT 2013-3280 to G.L.B.; PICT 2014-3658 to G.L.B. and H.E.G.; PICT 2013-1301 to H.E.G.; PICT 2015-1302 to A.J.F.-A.; PICT 2012-2493 to M.G.T., PICT 2018-01790 to M.G.T. and A.J.F.-A., all from Agencia Nacional de Promoción Cientıfica y Tecnolo ́ ́gica (ANPCyT; Argentina); PIP2011-205 (Consejo Nacional de Investigaciones Cientıficas y ́ Técnicas, CONICET, Argentina) to M.G.T.; an Alicia Moreau Chair from Paris Diderot University (France) to G.L.B. and A14S03 to G.L.B. and A.P. (MINCYTECOS SUD, Ministerio de Ciencia, Tecnologıa e Innovacio ́ ́n Productiva); grant 1112 from the Fondation ARC pour la Recherche sur le Cancer to A.P., and SAF2016- 75004-R (Ministerio de Economıa, Industria y Competitividad, Gobierno de Espan ́ ̃ a; MINECO, Spain) and PROMETEO/2018/055 (Generalitat Valenciana, Spain) to M.C. M.C. participates in COST Action CA15203 MITOEAGLE. A.J.F.-A., M.G.T., H.G. and G.L.B. are investigators from CONICET, G.L.B. and H.E.G. are professors at the University of Buenos Aires, Argentina; M.L.P., M.H., P.E.L.S., J.P. and A.A.C. received fellowships from CONICET, J.P. visited Carmo Fonseca’s Lab supported by H2020-Marie Sklodowska-Curie Research and Innovation Staff Exchanges [734825-LysoMod], A.P. is a professor at the University Paris Diderot.

Post-transcriptional regulation of gene expression plays a critical role in a variety of cellular processes during development. RNA binding proteins are fundamental mediators of post-transcriptional regulations that control mRNA expression by recognizing specific cis acting elements within the target transcripts. Smaug is a highly conserved sequence specific RNA-binding protein that is essential during Drosophila early embryogenesis. Smaug binds Smaug Recognition Elements (SRE) in the target mRNA and recruits additional factors, via protein-protein interactions, that regulate the bound mRNA. An emergent concept that signaling pathways can modulate RBP activity by post-translation modifications adds a new layer in the control of gene expression. During my thesis work, I sought to understand how the Hedgehog pathway regulates Smaug by promoting its phosphorylation. My work shows that HH signaling downregulates Smaug protein levels affecting its ability to repress mRNA translation. This negative effect seems to be dependent on the interaction between Smaug and the HH signal transducer Smoothened. Moreover, Smaug is constitutively phosphorylated in its RNA binding domain, which appears to be necessary for cytoplasmic Smaug foci formation., La régulation post-transcriptionnelle de l'expression génique joue un rôle essentiel dans divers processus cellulaires pendant le développement. Les protéines de liaison à l'ARN (RBP) sont des médiateurs fondamentaux des régulations post-transcriptionnelles qui contrôlent l'expression de l'ARNm en reconnaissant des séquences spécifiques dans les transcrits cibles. Smaug est une protéine de liaison à l'ARN conservée de la levure jusqu’à l’homme qui est essentielle pendant l'embryogenèse précoce de la drosophile. Smaug reconnaît et lie des éléments de reconnaissance de Smaug (SRE) dans ses ARNm cibles et recrute des facteurs supplémentaires, via des interactions protéine-protéine, qui régulent l'ARNm lié. Un concept qui émerge est celui des voies de signalisation pouvant moduler l'activité des RBP par des modifications post-traductionnelles, en ajoutant ainsi une couche supplémentaire dans le contrôle de l'expression des gènes.Au cours de mon travail de thèse, j'ai cherché à mettre en évidence que la voie de signalisation Hedgehog régule Smaug en favorisant sa phosphorylation. Mon travail montre que la signalisation HH diminue les niveaux de protéines Smaug affectant sa capacité à réprimer la traduction de l'ARNm. Cet effet négatif semble dépendre de l'interaction entre Smaug et le transducteur de signal HH, Smoothened. De plus, Smaug est constitutivement phosphorylée dans son domaine de liaison à l'ARN, ce qui semble être nécessaire pour la formation des foci cytoplasmiques de Smaug.

Post-transcriptional regulation of gene expression plays a critical role in a variety of cellular processes during development. RNA binding proteins are fundamental mediators of post-transcriptional regulations that control mRNA expression by recognizing specific cis acting elements within the target transcripts. Smaug is a highly conserved sequence specific RNA-binding protein that is essential during Drosophila early embryogenesis. Smaug binds Smaug Recognition Elements (SRE) in the target mRNA and recruits additional factors, via protein-protein interactions, that regulate the bound mRNA. An emergent concept that signaling pathways can modulate RBP activity by post-translation modifications adds a new layer in the control of gene expression. During my thesis work, I sought to understand how the Hedgehog pathway regulates Smaug by promoting its phosphorylation. My work shows that HH signaling downregulates Smaug protein levels affecting its ability to repress mRNA translation. This negative effect seems to be dependent on the interaction between Smaug and the HH signal transducer Smoothened. Moreover, Smaug is constitutively phosphorylated in its RNA binding domain, which appears to be necessary for cytoplasmic Smaug foci formation.; La régulation post-transcriptionnelle de l'expression génique joue un rôle essentiel dans divers processus cellulaires pendant le développement. Les protéines de liaison à l'ARN (RBP) sont des médiateurs fondamentaux des régulations post-transcriptionnelles qui contrôlent l'expression de l'ARNm en reconnaissant des séquences spécifiques dans les transcrits cibles. Smaug est une protéine de liaison à l'ARN conservée de la levure jusqu’à l’homme qui est essentielle pendant l'embryogenèse précoce de la drosophile. Smaug reconnaît et lie des éléments de reconnaissance de Smaug (SRE) dans ses ARNm cibles et recrute des facteurs supplémentaires, via des interactions protéine-protéine, qui régulent l'ARNm lié. Un concept qui émerge est celui des voies de signalisation pouvant moduler l'activité des RBP par des modifications post-traductionnelles, en ajoutant ainsi une couche supplémentaire dans le contrôle de l'expression des gènes.Au cours de mon travail de thèse, j'ai cherché à mettre en évidence que la voie de signalisation Hedgehog régule Smaug en favorisant sa phosphorylation. Mon travail montre que la signalisation HH diminue les niveaux de protéines Smaug affectant sa capacité à réprimer la traduction de l'ARNm. Cet effet négatif semble dépendre de l'interaction entre Smaug et le transducteur de signal HH, Smoothened. De plus, Smaug est constitutivement phosphorylée dans son domaine de liaison à l'ARN, ce qui semble être nécessaire pour la formation des foci cytoplasmiques de Smaug.