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Summary The mTERF gene family encodes for nucleic acid binding proteins that are predicted to regulate organellar gene expression in eukaryotes. Despite the implication of this gene family in plant development and response to abiotic stresses, a precise molecular function was assigned to only a handful number of its c. 30 members in plants.Using a reverse genetics approach in Arabidopsis thaliana and combining molecular and biochemical techniques, we revealed new functions for the chloroplast mTERF protein, MDA1.We demonstrated that MDA1 associates in vivo with components of the plastid‐encoded RNA polymerase and transcriptional active chromosome complexes. MDA1 protein binds in vivo and in vitro with specificity to 27‐bp DNA sequences near the 5′‐end of psbE and ndhA chloroplast genes to stimulate their transcription, and additionally promotes the stabilization of the 5′‐ends of processed psbE and ndhA messenger (m)RNAs. Finally, we provided evidence that MDA1 function in gene transcription likely coordinates RNA folding and the action of chloroplast RNA‐binding proteins on mRNA stabilization.Our results provide examples for the unexpected implication of DNA binding proteins and gene transcription in the regulation of mRNA stability in chloroplasts, blurring the boundaries between DNA and RNA metabolism in this organelle.

Background: The dystonias are a heterogeneous group of hyperkinetic disorders characterized by sustained or intermittent muscle contractions that cause abnormal movements and/or postures. Although more than 200 causal genes are known, many cases of primary dystonia have no clear genetic cause.Objectives: To identify the causal gene in a consanguineous family with three siblings affected by a complex persistent generalized dystonia, generalized epilepsy, and mild intellectual disability.Methods: We performed exome sequencing in the parents and two affected siblings and characterized the expression of the identified gene by immunohistochemistry in control human and zebrafish brains.Results: We identified a novel missense variant (c.142G>A (NM_032192); p.Glu48Lys) in the protein phosphatase 1 regulatory inhibitor subunit 1B gene (PPP1R1B) that was homozygous in all three siblings and heterozygous in the parents. This gene is also known as dopamine and cAMP-regulated neuronal phosphoprotein 32 (DARPP-32) and has been involved in the pathophysiology of abnormal movements. The uncovered variant is absent in public databases and modifies the conserved glutamate 48 localized close to the serine 45 phosphorylation site. The PPP1R1B protein was shown to be expressed in cells and regions involved in movement control, including projection neurons of the caudate-putamen, substantia nigra neuropil, and cerebellar Purkinje cells. The latter cells were also confirmed to be positive for PPP1R1B expression in the zebrafish brain.Conclusions: We report the association of a PPP1R1B/DARPP-32 variant with generalized dystonia in man. It might be relevant to include the sequencing of this new gene in the diagnosis of patients with otherwise unexplained movement disorders. © 2021 International Parkinson and Movement Disorder Society.

Proliferating cells experience a global reduction of transcription during mitosis, yet their cell identity is maintained and regulatory information is propagated from mother to daughter cells. Mitotic bookmarking by transcription factors has been proposed as a potential mechanism to ensure the reactivation of transcription at the proper set of genes exiting mitosis. Recently, mitotic transcription and waves of transcription reactivation have been observed in synchronized populations of human hepatoma cells. However, the study did not consider that mitotic-arrested cell populations progressively desynchronize leading to measurements of gene expression on a mixture of cells at different internal cell-cycle times. Moreover, it is not well understood yet what is the precise role of mitotic bookmarking on mitotic transcription as well as on the transcription reactivation waves. Ultimately, the core gene regulatory network driving the precise transcription reactivation dynamics remains to be identified. To address these questions, we developed a mathematical model to correct for the progressive desynchronization of cells and estimate gene expression dynamics with respect to a cell-cycle pseudotime. Furthermore, we used a multiple linear regression model to infer transcription factor activity dynamics. Our analysis allows us to characterize waves of transcription factor activities exiting mitosis and predict a core gene regulatory network responsible of the transcription reactivation dynamics. Moreover, we identified more than 60 transcription factors that are highly active during mitosis and represent new candidates of mitotic bookmarking factors which could be relevant therapeutic targets to control cell proliferation., Author summary Specific gene expression patterns confer particular identities to cells. During proliferation, cells undergo mitosis when chromosomes are formed and segregated into two new cells leading to a global downregulation of gene expression. Yet, cell identity is propagated from mother to daughter cells by the reactivation of gene expression at the appropriate set of genes once mitosis is completed. Mitotic bookmarking has been proposed as a mechanism to regulate this process. Indeed certain regulatory factors tag genes during mitosis to promote gene reactivation in the next cycle. Here we analyze gene expression over time measured on synchronized cell populations by using a new generation sequencing technique. To do so, we proposed a mathematical model to obtain the exact gene expression dynamics with respect to the cell-cycle progression and identified waves of genes reactivation during mitosis and the transition to the next cycle. Also, we developed a computational method that allowed us to predict key regulatory factors that drive this process and predict new candidates that could be involved in mitotic bookmarking. These regulatory factors could be relevant therapeutic targets to control cell proliferation.

The cause of Alzheimer’s disease (AD) is incompletely defined. To date, no mono-causal treatment has so far reached its primary clinical endpoints, probably due to the complexity and diverse neuropathology contributing to the neurodegenerative process. In the present paper, we describe the plausible etiological role of copper (Cu) imbalance in the disease. Cu imbalance is strongly associated with neurodegeneration in dementia, but a complete biochemical etiology consistent with the clinical, chemical, and genetic data is required to support a causative association, rather than just correlation with disease. We hypothesize that a Cu imbalance in the aging human brain evolves as a gradual shift from bound metal ion pools, associated with both loss of energy production and antioxidant function, to pools of loosely bound metal ions, involved in gain-of-function oxidative stress, a shift that may be aggravated by chemical aging. We explain how this may cause mitochondrial deficits, energy depletion of high-energy demanding neurons, and aggravated protein misfolding/oligomerization to produce different clinical consequences shaped by the severity of risk factors, additional comorbidities, and combinations with other types of pathology. Cu imbalance should be viewed and integrated with concomitant genetic risk factors, aging, metabolic abnormalities, energetic deficits, neuroinflammation, and the relation to tau, prion proteins, α-synuclein, TAR DNA binding protein-43 (TDP-43) as well as systemic comorbidity. Specifically, the Amyloid Hypothesis is strongly intertwined with Cu imbalance because amyloid-β protein precursor (AβPP)/Aβ are probable Cu/Zn binding proteins with a potential role as natural Cu/Zn buffering proteins (loss of function), and via the plausible pathogenic role of Cu-Aβ.

The RNA exosome is a key 3’−5’ exoribonuclease with an evolutionarily conserved structure and function. Its cytosolic functions require the co-factors SKI7 and the Ski complex. Here we demonstrate by co-purification experiments that the ARM-repeat protein RESURRECTION1 (RST1) and RST1 INTERACTING PROTEIN (RIPR) connect the cytosolic Arabidopsis RNA exosome to the Ski complex. rst1 and ripr mutants accumulate RNA quality control siRNAs (rqc-siRNAs) produced by the post-transcriptional gene silencing (PTGS) machinery when mRNA degradation is compromised. The small RNA populations observed in rst1 and ripr mutants are also detected in mutants lacking the RRP45B/CER7 core exosome subunit. Thus, molecular and genetic evidence supports a physical and functional link between RST1, RIPR and the RNA exosome. Our data reveal the existence of additional cytosolic exosome co-factors besides the known Ski subunits. RST1 is not restricted to plants, as homologues with a similar domain architecture but unknown function exist in animals, including humans., Cytosolic RNA degradation by the RNA exosome requires the Ski complex. Here the authors show that the proteins RST1 and RIPR assist the RNA exosome and the Ski complex in RNA degradation, thereby preventing the production of secondary siRNAs from endogenous mRNAs.

International audience; The Virtual Screening (VS) study described herein aimed at detecting novel Bromodomain BRD4 binders and relied on knowledge from public databases (ChEMBL, REAXYS) to establish a battery of predictive models of BRD activity for in silico selection of putative ligands. Beyond the actual discovery of new BRD ligands, this represented an opportunity to practically estimate the actual usefulness of public domain "Big Data" for robust predictive model building. Obtained models were used to virtually screen a collection of 2 million compounds from the Enamine company collection. This industrial partner then experimentally screened a subset of 2992 molecules selected by the VS procedure for their high likelihood to be active. Twenty nine confirmed hits were detected after experimental testing, representing 1% of the selected candidates. As a general conclusion, this study emphasizes once more that public structure-activity databases are nowadays key assets in drug discovery. Their usefulness is however limited by the state-of-the-art knowledge harvested so far by published studies. Target-specific structure activity information is rarely rich enough, and its heterogeneity makes it extremely difficult to exploit in rational drug design. Furthermore, published affinity measures serving to build models selecting compounds to be experimentally screened may not be well correlated with the experimental hit selection criterion (in practice, often imposed by equipment constraints). Nevertheless, a robust 2.6-fold increase in hit rate with respect to an equivalent, random screening campaign showed that machine learning is able to extract some real knowledge in spite of all the noise in structure-activity data.

Post-transcriptional modification of tRNA wobble adenosine into inosine is crucial for decoding multiple mRNA codons by a single tRNA. The eukaryotic wobble adenosine-to-inosine modification is catalysed by the ADAT (ADAT2/ADAT3) complex that modifies up to eight tRNAs, requiring a full tRNA for activity. Yet, ADAT catalytic mechanism and its implication in neurodevelopmental disorders remain poorly understood. Here, we have characterized mouse ADAT and provide the molecular basis for tRNAs deamination by ADAT2 as well as ADAT3 inactivation by loss of catalytic and tRNA-binding determinants. We show that tRNA binding and deamination can vary depending on the cognate tRNA but absolutely rely on the eukaryote-specific ADAT3 N-terminal domain. This domain can rotate with respect to the ADAT catalytic domain to present and position the tRNA anticodon-stem-loop correctly in ADAT2 active site. A founder mutation in the ADAT3 N-terminal domain, which causes intellectual disability, does not affect tRNA binding despite the structural changes it induces but most likely hinders optimal presentation of the tRNA anticodon-stem-loop to ADAT2., Graphical Abstract Graphical abstractADAT catalyses tRNAs wobble adenosine-to-inosine modification through ADAT3 recognition of tRNAs 3D structure and anticodon-loop presentation to ADAT2 catalytic subunit. ADAT3 V128M mutation hampers presentation, leading to neurodevelopmental disorders.

Using coevolution network interference based on comparison of two phylogenetically distantly related isolates, one from the main group M and the other from the minor group O of HIV-1, we identify, in the C-terminal domain (CTD) of integrase, a new functional motif constituted by four noncontiguous amino acids (N(222)K(240)N(254)K(273)). Mutating the lysines abolishes integration through decreased 3′ processing and inefficient nuclear import of reverse-transcribed genomes. Solution of the crystal structures of wild-type (wt) and mutated CTDs shows that the motif generates a positive surface potential that is important for integration. The number of charges in the motif appears more crucial than their position within the motif. Indeed, the positions of the K’s could be permutated or additional K’s could be inserted in the motif, generally without affecting integration per se. Despite this potential genetic flexibility, the NKNK arrangement is strictly conserved in natural sequences, indicative of an effective purifying selection exerted at steps other than integration. Accordingly, reverse transcription was reduced even in the mutants that retained wt integration levels, indicating that specifically the wt sequence is optimal for carrying out the multiple functions that integrase exerts. We propose that the existence of several amino acid arrangements within the motif, with comparable efficiencies of integration per se, might have constituted an asset for the acquisition of additional functions during viral evolution. IMPORTANCE Intensive studies of HIV-1 have revealed its extraordinary ability to adapt to environmental and immunological challenges, an ability that is also at the basis of antiviral treatment escape. Here, by deconvoluting the different roles of the viral integrase in the various steps of the infectious cycle, we report how the existence of alternative equally efficient structural arrangements for carrying out one function opens up the possibility of adapting to the optimization of further functionalities exerted by the same protein. Such a property provides an asset to increase the efficiency of the infectious process. On the other hand, though, the identification of this new motif provides a potential target for interfering simultaneously with multiple functions of the protein.

The liver zonation is an important phenomenon characterized by a gradient of several functions along the liver acinus. However, this gradient remains difficult to reproduce in in-vitro conditions, making the obtention of an in-vitro method to recapitulate the liver zonation a challenging issue. In this study, we evaluated the spatial evolution of the transcriptome profile of human induced pluripotent stem cells (hiPSCs) differentiated toward hepatocytes-like cells (HLCs) phenotype in a microfluidic biochip environment. Cells collected at the inlet of the biochip, where the oxygen concentration is higher, were identified by the expression of genes involved in metabolic pathways related to cellular reorganization and cell proliferation. Cells collected in the middle and at the outlet of the biochips, where oxygen concentrations are lower, were characterized by the upregulation of genes involved in cellular detoxification processes (CYP450), PPAR signaling or arginine biosynthesis. A subset of 16 transcription factors (TFs) was extracted and identified as upstream regulators to HNF1A and PPARA. These TFs are also known as regulators to target genes engaged in the Wnt/βcatenin pathway, in the TGFβ/BMP/SMAD signaling, in the transition between epithelial mesenchymal transition (EMT) and mesenchymal epithelial transition (MET), in the homeostasis of lipids, bile acids and carbohydrates homeostasis, in drug metabolism, in the estrogen processing and in the oxidative stress response. Overall, the analysis allowed to confirm a partial zonation-like pattern in hiPSCs-derived HLCs in the microfluidic biochip environment. These results provide important insights into the reproduction of liver zonation in-vitro for a better understanding of the phenomenon.

Translation fidelity relies essentially on the ability of ribosomes to accurately recognize triplet interactions between codons on mRNAs and anticodons of tRNAs. To determine the codon-anticodon pairs that are efficiently accepted by the eukaryotic ribosome, we took advantage of the IRES from the intergenic region (IGR) of the Cricket Paralysis Virus. It contains an essential pseudoknot PKI that structurally and functionally mimics a codon-anticodon helix. We screened the entire set of 4096 possible combinations using ultrahigh-throughput screenings combining coupled transcription/translation and droplet-based microfluidics. Only 97 combinations are efficiently accepted and accommodated for translocation and further elongation: 38 combinations involve cognate recognition with Watson-Crick pairs and 59 involve near-cognate recognition pairs with at least one mismatch. More than half of the near-cognate combinations (36/59) contain a G at the first position of the anticodon (numbered 34 of tRNA). G34-containing tRNAs decoding 4-codon boxes are almost absent from eukaryotic genomes in contrast to bacterial genomes. We reconstructed these missing tRNAs and could demonstrate that these tRNAs are toxic to cells due to their miscoding capacity in eukaryotic translation systems. We also show that the nature of the purine at position 34 is correlated with the nucleotides present at 32 and 38.

Many RNA-RNA interactions depend on molecular chaperones to form and remain stable in living cells. A prime example is the RNA chaperone Hfq, which is a critical effector involved in regulatory interactions between small RNAs (sRNAs) and cognate target mRNAs in Enterobacteriaceae. While there is a great deal of in vitro biochemical evidence supporting the model that Hfq enhances rates or affinities of sRNA:mRNA interactions, there is little corroborating in vivo evidence. Here we used in vivo tools including reporter genes, co-purification assays, and super-resolution microscopy to analyze the role of Hfq in RyhB-mediated regulation, and we found that Hfq is often unnecessary for efficient RyhB:mRNA complex formation in vivo. Remarkably, our data suggest that a primary function of Hfq is to promote RyhB-induced cleavage of mRNA targets by RNase E. Moreover, our work indicates that Hfq plays a more limited role in dictating regulatory outcomes following sRNAs RybB and DsrA complex formation with specific target mRNAs. Our investigation helps evaluate the roles played by Hfq in some RNA-mediated regulation.

The view from the south is, more than ever, dominated by ominous signs of change. Antarctica and the Southern Ocean are intrinsic to the Earth system, and their evolution is intertwined with and influences the course of the Anthropocene. In turn, changes in the Antarctic affect and presage humanity's future. Growing understanding is countering popular beliefs that Antarctica is pristine, stable, isolated, and reliably frozen. An aspirational roadmap for Antarctic science has facilitated research since 2014. A renewed commitment to gathering further knowledge will quicken the pace of understanding of Earth systems and beyond. Progress is already evident, such as addressing uncertainties in the causes and pace of ice loss and global sea-level rise. However, much remains to be learned. As an iconic global “commons,” the rapidity of Antarctic change will provoke further political action. Antarctic research is more vital than ever to a sustainable future for this One Earth.

Grapevine fanleaf virus (GFLV) is a picorna-like plant virus transmitted by nematodes that affects vineyards worldwide. Nanobody (Nb)-mediated resistance against GFLV has been created recently and shown to be highly effective in plants including grapevine, but the underlying mechanism is unknown. Here we present the high-resolution cryo-EM structure of the GFLV-Nb23 complex which provides the basis for the molecular recognition by the nanobody. The structure reveals a composite binding site bridging over 3 domains of the capsid protein (CP) monomer. The structure provides a precise mapping of the Nb23 epitope on the GFLV capsid in which the antigen loop is accommodated through an induced fit mechanism. Moreover, we uncover and characterize several resistance-breaking GFLV isolates with amino acids mapping within this epitope, including C-terminal extensions of the CP, which would sterically interfere with Nb binding. Escape variants with such extended CP fail to be transmitted by nematodes linking Nb-mediated resistance to vector transmission. Together, these data provide insights into the molecular mechanism of Nb23-mediated recognition of GFLV and of virus resistance loss.SignificanceGrapevine fanleaf virus (GFLV) is a picorna-like plant virus that severely impacts vineyards worldwide. While Nanobodies (Nb) confer resistance to GFLV in plants the underlying molecular mechanism of action is unknown. Here we present the high-resolution cryo-EM structure of the GFLV-Nb complex. It uncovers the conformational epitope on the capsid surface which is a composite binding site into which the antigen loop is accommodated through an induced fit mechanism. Furthermore, we describe several resistance-breaking isolates of GFLV with reduced Nb binding capacity. Those that carry a C-terminal extension also fail to be transmitted by nematodes. Together, these data provide structure-function insights into the Nb-GFLV recognition and the molecular mechanism leading to loss of resistance.

Gametes are generated through a specialized cell differentiation process, meiosis which, in most mammals, is initiated in ovaries during fetal life. It is widely admitted that all-transretinoic acid (ATRA) is the molecular signal triggering meiosis initiation in mouse female germ cells, but a genetic approach in which ATRA synthesis is impaired disputes this proposal. In the present study, we investigated the contribution of endogenous ATRA to meiosis by analyzing fetuses lacking all RARs ubiquitously, obtained through a tamoxifen-inducible cre recombinase-mediated gene targeting approach. Efficient ablation of RAR-coding genes was assessed by the multiple congenital abnormalities displayed by the mutant fetuses. Unexpectedly, their germ cells robustly expressed STRA8, REC8, SYCP1 and SYCP3, showing that RAR are actually dispensable up to the zygotene stage of meiotic prophase I. Thus our study goes against the current model according to which meiosis is triggered by endogenous ATRA in the developing ovary and revives the identification of the meiosis-preventing substance synthesized by CYP26B1 in the fetal testis.

Camera images and video recordings are simple and non-invasive tools to investigate animals in their natural habitat. Quantitative evaluations, however, often require an exact reconstruction of object positions, sizes, and distances in the image. Here, we provide an open source software package to perform such calculations. Our approach allows the user to correct for perspective distortion, transform images to “bird’s-eye” view projections, or transform image-coordinates to real-world coordinates and vice versa. The extrinsic camera parameters that are necessary to perform such image corrections and transformations (elevation, tilt/roll angle, and heading of the camera) are obtained from the image using contextual information such as a visible horizon, GPS coordinates of landmarks, known object sizes, or images of the same object obtained from different viewing angles. All mathematical operations are implemented in the Python package CameraTransform. The performance of the implementation is evaluated using computer-generated synthetic images with known camera parameters. Moreover, we test our algorithm on images of emperor penguin colonies, and demonstrate that the camera tilt and roll angles can be estimated with an error of less than one degree, and the camera elevation with an error of less than 5%. The CameraTransform software package simplifies camera matrix-based image transformations and the extraction of quantitative image information. An extensive documentation and usage examples in an ecological context are provided at http://cameratransform.readthedocs.io. Keywords: Perspective projection, Quantitative image analysis, Geo-referencing, Camera lens distortions

The farnesoid-X-receptorα (FXRα; NR1H4) is one of the main bile acid (BA) receptors. During the last decades, through the use of pharmalogical approaches and transgenic mouse models, it has been demonstrated that the nuclear receptor FXRα controls numerous physiological functions such as glucose or energy metabolisms. It is also involved in the etiology or the development of several pathologies. Here, we will review the unexpected roles of FXRα on the male reproductive tract. FXRα has been demonstrated to play functions in the regulation of testicular and prostate homeostasis. Even though additional studies are needed to confirm these findings in humans, the reviewed reports open new field of research to better define the effects of bile acid-FXRα signaling pathways on fertility disorders and cancers.

Recent outbreaks of dangerous viral infections, such as Ebola virus disease, Zika fever, etc., are forcing the search for new antiviral compounds. Preferably, such compounds should possess broad-spectrum antiviral activity, as the development of drugs for the treatment of dozens of viral infections lacking specific treatment would require significant resources. Antiviral activity data present in public resources are very sparse and further investigation of structure-activity relationships is necessary. One of the strategies could be the investigation of chemical space around known active compounds and assessment of activity against closely related viruses in order to fill in the antiviral activity matrix. Here we present an investigation of antiviral activity using universal maps built with generative topographic mapping (GTM) algorithm. The GTM-based maps were used to find commercially available compounds in close proximity to already known compounds with anti-flaviviral and anti-enteroviral activities. Selected compounds were then assessed in cell-based assays against tick-borne encephalitis virus (TBEV) and a panel of enteroviruses. This approach allowed us to identify 23 new compounds showing anti-TBEV activity with EC50 values in micromolar and submicromolar range.

The previously reported procedure to generate "universal" Generative Topographic Maps (GTMs) of the drug-like chemical space is in practice a multi-task learning process, in which both operational GTM parameters (example: map grid size) and hyperparameters (key example: the molecular descriptor space to be used) are being chosen by an evolutionary process in order to fit/select "universal" GTM manifolds. After selection (a one-time task aimed at optimizing the compromise in terms of neighborhood behavior compliance, over a large pool of various biological targets), for any further use the manifolds are ready to provide "fit-free" predictive models. Using any structure-activity set-irrespectively whether the associated target served at map fitting stage or not-the generation or "coloring" a property landscape enables predicting the property for any external molecule, with zero additional fitable parameters involved. While previous works have signaled the excellent behavior of such models in aggressive three-fold cross-validation assessments of their predictive power, the present work wished to explore their behavior in Virtual Screening (VS), here simulated on hand of external DUD ligand and decoy series that are fully disjoint from the ChEMBL-extracted landscape coloring sets. Beyond the rather robust results of the universal GTM manifolds in this challenge, it could be shown that the descriptor spaces selected by the evolutionary multi-task learner were intrinsically able to serve as an excellent support for many other VS procedures, starting from parameter-free similarity searching, to local (target-specific) GTM models, to parameter-rich, nonlinear Random Forest and Neural Network approaches.

Hbs1 has been established as a central component of the cell’s translational quality control pathways in both yeast and prokaryotic models; however, the functional characteristics of its human ortholog (Hbs1L) have not been well-defined. We recently reported a novel human phenotype resulting from a mutation in the critical coding region of the HBS1L gene characterized by facial dysmorphism, severe growth restriction, axial hypotonia, global developmental delay and retinal pigmentary deposits. Here we further characterize downstream effects of the human HBS1L mutation. HBS1L has three transcripts in humans, and RT-PCR demonstrated reduced mRNA levels corresponding with transcripts V1 and V2 whereas V3 expression was unchanged. Western blot analyses revealed Hbs1L protein was absent in the patient cells. Additionally, polysome profiling revealed an abnormal aggregation of 80S monosomes in patient cells under baseline conditions. RNA and ribosomal sequencing demonstrated an increased translation efficiency of ribosomal RNA in Hbs1L-deficient fibroblasts, suggesting that there may be a compensatory increase in ribosome translation to accommodate the increased 80S monosome levels. This enhanced translation was accompanied by upregulation of mTOR and 4-EBP protein expression, suggesting an mTOR-dependent phenomenon. Furthermore, lack of Hbs1L caused depletion of Pelota protein in both patient cells and mouse tissues, while PELO mRNA levels were unaffected. Inhibition of proteasomal function partially restored Pelota expression in human Hbs1L-deficient cells. We also describe a mouse model harboring a knockdown mutation in the murine Hbs1l gene that shared several of the phenotypic elements observed in the Hbs1L-deficient human including facial dysmorphism, growth restriction and retinal deposits. The Hbs1lKO mice similarly demonstrate diminished Pelota levels that were rescued by proteasome inhibition., Author summary We have identified a patient with biallelic mutations in HBS1L affecting Hbs1LV1 and V2 transcripts leading to a loss of Hbs1L implicated in ribosomal recycling. In contrast to yeast studies, loss of Hbs1LV1/V2 in human cells did not appear to impact the translational quality control mechanisms of non-stop and no-go decay. However, patient cells demonstrated accumulation of free 80S ribosomes based on polysome profiling. In addition, Hbs1LV1/V2 deficient cells demonstrated an increase in translation efficiency of ribosomal mRNA based on RiboSeq data, which may suggest an attempt to compensate for defective mobilization of free 80S ribosomes. The patient samples demonstrated increased 4EBP1 and mTOR expression and phosphorylation compared to controls, suggesting an mTOR-dependent ribosomal RNA regulation is involved in the response to Hbs1LV1/V2 deficiency. Loss of Hbs1L in both human and mouse fibroblasts lead to diminished Pelota levels, and this phenomenon could be partially rescued by proteasome inhibition. In all, these data support a role for Hbs1LV1/V2 as a Pelota binding partner with a specific function in utilization of free ribosomes.

International audience; Background: Platinum (II)-based drugs are widely used chemother-apeutic agents. However, as they have many side effects, numerous platinum (Pt) derivatives have been developed during the last years. Furthermore, some cancer treatments including Pt compounds are able to activate the antitumor immune response by inducing a particular cell death: the immunogenic cell death (ICD). This process is characterized by the exposure of the endoplasmic reticulum chaperone calreticulin (CRT) at the cell surface as well as the release of ATP and non-histone chromatin-binding protein high mobility group box 1 (HMGB1) which serve as immunostimulatory damage-associated molecular patterns (DAMPs) and increase the antitumor immune response. Based on these considerations, we decided to focus on N-het-erocyclic carbene Pt complexes associated with the transfection agent polyethylenimine (PEI) to create multivalent cationic platinum compounds (NHC-Pt(II)-PEI). Materials and methods: We investigated the cytotoxic activity of our derivatives in vitro by flow cytometry and in vivo in different mouse models. Then, we evaluated by an IFN-g ELISpot assay the potential implication of the immune response on the NHC-Pt(II)-PEI in vivo effect in immunocompetent mice bearing tumors treated with Pt particles. Moreover, we assessed if NHC-Pt(II)-PEI were able to induce ICD by analysing the exposure of CRT at the cell surface by flow cytometry. We also combined our Pt derivative with immune danger signals, like poly-inosinic: polycytidylic acid (polyI:C), to enhance this effect. Results: Our conjugate displayed a good cytotoxic activity in vitro on several cancer cell lines as well as an antitumor effect in vivo in immunodeficient [1] and immunocompetent mice, in the same range than clinical used Pt drugs, but with fewer side effects. First results showed exposure of CRT at the cell surface of cancer cells treated with NHC-Pt(II)-PEI, suggesting the induction of ICD. To improve the antitumor immune response, we associated NHC-Pt(II)-PEI with polyI:C and found that both compounds formed a complex and we are now evaluating its cytotoxicity and effect on the immune response. Conclusions: Altogether our results reveal the possibility of creating Pt derivatives that can be used as chemotherapeutic agents by killing tumor cells and as immunotherapeutic agents by triggering the anti-tumor immune response.

International audience; A set of variously substituted aurones was synthesized and evaluated against Methicillin-Resistant S. aureus (MRSA) and P. aeruginosa. Several analogues were found active against MRSA, but no effect was recorded against P. aeruginosa. Compounds 27, 30 and 33 showed low cytotoxicity, and were tested against a full range of bacterial (Gram-positive and Gram-negative) and fungal species, including resistant strains. These aurones displayed a selective inhibition of Gram-positive bacteria with excellent Therapeutic Index values, while showing no significant action on several Gram-negative strains, H. pylori and V. alginolyticus being the only susceptible strains among the Gram-negative bacteria tested. A per-meabilization assay showed that the antibacterial activity of at least some of the aurones could be linked to alterations of the bacterial membrane. Overall, this study endorses the use of the aurone scaffold for the development of new potent and selective antibacterial agents.

This paper reports SVR (Support Vector Regression) and GTM (Generative Topographic Mapping) modeling of three kinetic properties of cycloaddition reactions: rate constant (logk), activation energy (Ea) and pre-exponential factor (logA). A data set of 1849 reactions, comprising (4+2), (3+2) and (2+2) cycloadditions (CA) were studied in different solvents and at different temperatures. The reactions were encoded by the ISIDA fragment descriptors generated for Condensed Graph of Reaction (CGR). For a given reaction, a CGR condenses structures of all the reactants and products into one single molecular graph, described both by conventional chemical bonds and "dynamical" bonds characterizing chemical transformations. Different scenarios of logk assessment were exploited: direct modeling, application of the Arrhenius equation and temperature-scaled GTM landscapes. The logk models with optimal cross-validated statistics (Q2 =0.78-0.94 RMSE=0.45-0.86) have been challenged to predict rates for the external test set of 200 reactions, comprising both reactions that were not present in the training set, and training set transformations performed under different reaction conditions. The models are freely available on our web-server: http://cimm.kpfu.ru/models.

Many animal and plant viruses rely on vectors for their transmission from host to host. Grapevine fanleaf virus (GFLV), a picorna-like virus from plants, is transmitted specifically by the ectoparasitic nematode Xiphinema index. The icosahedral capsid of GFLV, which consists of 60 identical coat protein subunits (CP), carries the determinants of this specificity. Here, we provide novel insight into GFLV transmission by nematodes through a comparative structural and functional analysis of two GFLV variants. We isolated a mutant GFLV strain (GFLV-TD) poorly transmissible by nematodes, and showed that the transmission defect is due to a glycine to aspartate mutation at position 297 (Gly297Asp) in the CP. We next determined the crystal structures of the wild-type GFLV strain F13 at 3.0 Å and of GFLV-TD at 2.7 Å resolution. The Gly297Asp mutation mapped to an exposed loop at the outer surface of the capsid and did not affect the conformation of the assembled capsid, nor of individual CP molecules. The loop is part of a positively charged pocket that includes a previously identified determinant of transmission. We propose that this pocket is a ligand-binding site with essential function in GFLV transmission by X. index. Our data suggest that perturbation of the electrostatic landscape of this pocket affects the interaction of the virion with specific receptors of the nematode's feeding apparatus, and thereby severely diminishes its transmission efficiency. These data provide a first structural insight into the interactions between a plant virus and a nematode vector.

International audience

International audience; X-linked myotubular myopathy (XLMTM) is a fatal pediatric disease where affected boys display profound weakness of the skeletal muscles. Possible therapies are under development but robust outcome measures in animal models are required for effective translation to human patients. We established a naturally-occurring canine model, where XLMTM dogs display clinical symptoms similar to those observed in humans. The aim of this study was to determine potential endpoints for the assessment of future treatments in this model. Video-based gait analysis was selected, as it is a well-established method of assessing limb function in neuromuscular disease and measures have been correlated to the patient's quality of life. XLMTM dogs (N = 3) and their true littermate wild type controls (N = 3) were assessed at 4-5 time points, beginning at 10 weeks and continuing through 17 weeks. Motion capture and an instrumented carpet were used separately to evaluate spatiotemporal and kinematic changes over time. XLMTM dogs walk more slowly and with shorter stride lengths than wild type dogs, and these differences became greater over time. However, there was no clear difference in angular measures between affected and unaffected dogs. These data demonstrate that spatiotemporal parameters capture functional changes in gait in an XLMTM canine model and support their utility in future therapeutic trials.

International audience; Vitamin A is instrumental to mammalian reproduction. Its metabolite, retinoic acid (RA), acts in a hormone-like manner through binding to and activating three nuclear receptor isotypes, RA receptor (RAR)α (RARA), RARβ, and RARγ (RARG). Here, we show that 1) RARG is expressed by A aligned (Aal) spermatogonia, as well as during the transition from Aal to A1 spermatogonia, which is known to require RA; and 2) ablation of Rarg, either in the whole mouse or specifically in spermatogonia, does not affect meiosis and spermiogenesis but impairs the Aal to A1 transition in the course of some of the seminiferous epithelium cycles. Upon ageing, this phenomenon yields seminiferous tubules containing only spermatogonia and Sertoli cells. Altogether, our findings indicate that RARG cell-autonomously transduces, in undifferentiated spermatogonia of adult testes, a RA signal critical for spermatogenesis. During the prepubertal spermatogenic wave, the loss of RARG function can however be compensated by RARA, as indicated by the normal timing of appearance of meiotic cells in Rarg-null testes. Accordingly, RARG- and RARA-selective agonists are both able to stimulate Stra8 expression in wild-type prepubertal testes. Interestingly, inactivation of Rarg does not impair expression of the spermatogonia differentiation markers Kit and Stra8, contrary to vitamin A deficiency. This latter observation supports the notion that the RA-signaling pathway previously shown to operate in Sertoli cells also participates in spermatogonia differentiation.

International audience; 18th International Congress of Developmental Biology, Natil Univ Singapore, Singapore, June 2017

International audience; The influenza A virus genome consists of eight viral RNAs (vRNAs) that form viral ribonucleoproteins (vRNPs). Even though evidence supporting segment-specific packaging of vRNAs is accumulating, the mechanism ensuring selective packaging of one copy of each vRNA into the viral particles remains largely unknown. We used electron tomography to show that the eight vRNPs emerge from a common 'transition zone' located underneath the matrix layer at the budding tip of the virions, where they appear to be interconnected and often form a star-like structure. This zone appears as a platform in 3D surface rendering and is thick enough to contain all known packaging signals. In vitro, all vRNA segments are involved in a single network of intermolecular interactions. The regions involved in the strongest interactions were identified and correspond to known packaging signals. A limited set of nucleotides in the 5' region of vRNA 7 was shown to interact with vRNA 6 and to be crucial for packaging of the former vRNA. Collectively, our findings support a model in which the eight genomic RNA segments are selected and packaged as an organized supramolecular complex held together by direct base pairing of the packaging signals.

The thermophilic bacterial laccase CotA from Bacillus subtilis adsorbed on graphite electrodes enables monitoring of the temperature dependent direct biocatalytic O2 reduction. Its entrapment in two different Os-complex modified redox hydrogels is the basis for mediated bioelectroreduction of O2. Besides the temperature and pH dependence of the bioelectrocatalytic response chloride and fluoride inhibition studies demonstrate that the Os-complexes are bound to the T1 copper centre of the enzyme. The interaction between CotA and the Os-complex modified polymers prevents inhibition by chloride ions.

Chantier qualité GA; International audience; Sialyltransferases are key enzymes in the biosynthesis of sialoglycoconjugates that catalyze the transfer of sialic residue from its activated form to an oligosaccharidic acceptor. β-Galactoside α2,6-sialyltransferases ST6Gal I and ST6Gal II are the two unique members of the ST6Gal family described in higher vertebrates. The availability of genome sequences enabled the identification of more distantly related invertebrates' st6gal gene sequences and allowed us to propose a scenario of their evolution. Using a phylogenomic approach, we present further evidence of an accelerated evolution of the st6gal1 genes both in their genomic regulatory sequences and in their coding sequence in reptiles, birds, and mammals known as amniotes, whereas st6gal2 genes conserve an ancestral profile of expression throughout vertebrate evolution.

Retroviruses require both spliced and unspliced RNAs for replication. Accumulation of Rous Sarcoma virus (RSV) unspliced RNA depends upon the negative regulator of splicing (NRS). Its 5'-part is considered as an ESE binding SR proteins. Its 3'-part contains a decoy 5'-splice site (ss), which inhibits splicing at the bona fide 5'-ss. Only the 3D structure of a small NRS fragment had been experimentally studied. Here, by chemical and enzymatic probing, we determine the 2D structure of the entire RSV NRS. Structural analysis of other avian NRSs and comparison with all sequenced avian NRSs is in favour of a phylogenetic conservation of the NRS 2D structure. By combination of approaches: (i) in vitro and in cellulo splicing assays, (ii) footprinting assays and (iii) purification and analysis of reconstituted RNP complex, we define a small NRS element retaining splicing inhibitory property. We also demonstrate the capability of the SR protein 9G8 to increase NRS activity in vitro and in cellulo. Altogether these data bring new insights on how NRS fine tune splicing activity.

International audience; The HIV-1 viral infectivity factor (Vif) allows productive infection of non-permissive cells (including most natural HIV-1 targets) by counteracting the cellular cytosine deaminases APOBEC-3G (hA3G) and hA3F. The Vif-induced degradation of these restriction factors by the proteasome has been extensively studied, but little is known about the translational repression of hA3G and hA3F by Vif, which has also been proposed to participate in Vif function. Here, we studied Vif binding to hA3G mRNA and its role in translational repression. Filter binding assays and fluorescence titration curves revealed that Vif tightly binds to hA3G mRNA. Vif overall binding affinity was higher for the 3'UTR than for the 5'UTR, even though this region contained at least one high affinity Vif binding site (apparent K(d) = 27 +/- 6 nM). Several Vif binding sites were identified in 5' and 3'UTRs using RNase footprinting. In vitro translation evidenced that Vif inhibited hA3G translation by two mechanisms: a main time-independent process requiring the 5'UTR and an additional time-dependent, UTR-independent process. Results using a Vif protein mutated in the multimerization domain suggested that the molecular mechanism of translational control is more complicated than a simple physical blockage of scanning ribosomes.

Systematic investigation of a large number of trial rigid-body refinements leads to an optimized multiple-zone protocol with a larger convergence radius., Rigid-body refinement is the constrained coordinate refinement of one or more groups of atoms that each move (rotate and translate) as a single body. The goal of this work was to establish an automatic procedure for rigid-body refinement which implements a practical compromise between runtime requirements and convergence radius. This has been achieved by analysis of a large number of trial refinements for 12 classes of random rigid-body displacements (that differ in magnitude of introduced errors), using both least-squares and maximum-likelihood target functions. The results of these tests led to a multiple-zone protocol. The final parameterization of this protocol was optimized empirically on the basis of a second large set of test refinements. This multiple-zone protocol is implemented as part of the phenix.refine program.

MicroRNAs (miRNAs) have emerged as key players in the regulation of expression of target mRNAs expression. They have been associated with diverse biological processes, and recent studies have demonstrated that miRNAs play a role in inflammatory responses. We reported previously that LPS-activated fibroblast-like synoviocytes (FLS) isolated from rheumatoid arthritis (RA) patients express IL-18 mRNA but they do not release IL-18. Based on the observation that this inhibition was due to a rapid degradation of IL-18 mRNA, our group has conducted a study to identify miRNAs that could play a role in the “antiinflammatory” response of LPS-activated RA FLS. LPS challenge modulated the expression of 63 miRNAs as assessed by microarray analysis. Fifteen miRNAs were up-regulated, including miR-346, for which overexpression upon LPS treatment was validated by quantitative RT-PCR. We then transfected FLS with an antisense oligonucleotide targeting miR-346 and found that, in these conditions, IL-18 release could be measured upon LPS activation of FLS. Moreover, we also demonstrated that miR-346 indirectly regulated IL-18 release by indirectly inhibiting LPS-induced Bruton’s tyrosine kinase expression in LPS-activated RA FLS. These findings suggest that miRNAs function as regulators that help to fine-tune the inflammatory response in RA.

EGFR or ERBB2 contributes to prostate cancer (PCa) progression by activating the androgen receptor (AR) in hormone-poor conditions. Here, we investigated the mechanisms by which androgens regulate EGFR and ERBB2 expression in PCa cells. In steroid-depleted medium (SDM), EGFR protein was less abundant in androgen-sensitive LNCaP than in androgen ablation–resistant 22Rv1 cells, whereas transcript levels were similar. Dihydrotestosterone (DHT) treatment increased both EGFR mRNA and protein levels and stimulated RNA polymerase II recruitment to the EGFR gene promoter, whereas it decreased ERBB2 transcript and protein levels in LNCaP cells. DHT altered neither EGFR or ERBB2 levels nor the abundance of prostate-specific antigen (PSA), TMEPA1, or TMPRSS2 mRNAs in 22Rv1 cells, which express the full-length and a shorter AR isoform deleted from the COOH-terminal domain (ARΔCTD). The contribution of both AR isoforms to the expression of these genes was assessed by small interfering RNAs targeting only the full-length or both AR isoforms. Silencing of both isoforms strongly reduced PSA, TMEPA1, and TMPRSS2 transcript levels. Inhibition of both AR isoforms did not affect EGFR and ERBB2 transcript levels but decreased EGFR and increased ERBB2 protein levels. Proliferation of 22Rv1 cells in SDM was inhibited in the absence of AR and ARΔCTD. A further decrease was obtained with PKI166, an EGFR/ERBB2 kinase inhibitor. Overall, we showed that ARΔCTD is responsible for constitutive EGFR expression and ERBB2 repression in 22Rv1 cells and that ARΔCTD and tyrosine kinase receptors are necessary for sustained 22Rv1 cell growth. [Cancer Res 2009;69(7):2941–9]

International audience; X-rays interact with biological matter and cause damage. Proteins and other macromolecules are damaged primarily by ionizing X-ray photons and secondarily by reactive radiolytic chemical species. In particular, protein molecules are damaged during X-ray diffraction experiments with protein crystals, which is, in many cases, a serious hindrance to structure solution. The local X-ray-induced structural changes of the protein molecule have been studied using a number of model systems. However, it is still not well understood whether these local chemical changes lead to global structural changes in protein and what the mechanism is. We present experimental evidence at atomic resolution indicating the movement of large parts of the protein globule together with bound water molecules in the early stages of radiation damage to the protein crystal. The data were obtained from a crystal cryocooled to approximately 100 K and diffracting to 1 A. The movement of the protein structural elements occurs simultaneously with the decarboxylation of several glutamate and aspartate residues that mediate contacts between moving protein structural elements and with the rearrangement of the water network. The analysis of the anisotropy of atomic displacement parameters reveals that the observed atomic movements occur at different rates in different unit cells of the crystal. Thus, the examination of the cooperative atomic movement enables us to better understand how radiation-induced local chemical and structural changes of the protein molecule eventually lead to disorder in protein crystals.

International audience; DNA repair and maintenance of genome stability are crucial to cellular and organismal function, and defects in these processes have been implicated in cancer and ageing. Detailed molecular, biochemical and genetic analyses have outlined the molecular framework involved in cellular DNA-repair pathways, but recent cell-biological approaches have revealed important roles for the spatial and temporal organization of the DNA-repair machinery during the recognition of DNA lesions and the assembly of repair complexes. It has also become clear that local higher-order chromatin structure, chromatin dynamics and non-random global genome organization are key factors in genome maintenance. These cell-biological features of DNA repair illustrate an emerging role for nuclear architecture in multiple aspects of genome maintenance.

International audience; Cyp26b1 encodes a cytochrome-P450 enzyme that catabolizes retinoic acid (RA), a vitamin A derived signaling molecule. We have examined Cyp26b1(-/-) mice and report that mutants exhibit numerous abnormalities in cranial neural crest cell derived tissues. At embryonic day (E) 18.5 Cyp26b1(-/-) animals exhibit a truncated mandible, abnormal tooth buds, reduced ossification of calvaria, and are missing structures of the maxilla and nasal process. Some of these abnormalities may be due to defects in formation of Meckel's cartilage, which is truncated with an unfused distal region at E14.5 in mutant animals. Despite the severe malformations, we did not detect any abnormalities in rhombomere segmentation, or in patterning and migration of anterior hindbrain derived neural crest cells. Abnormal migration of neural crest cells toward the posterior branchial arches was observed, which may underlie defects in larynx and hyoid development. These data suggest different periods of sensitivity of anterior and posterior hindbrain neural crest derivatives to elevated levels of RA in the absence of CYP26B1. Developmental Dynamics 238:732-745, 2009. (c) 2009 Wiley-Liss, Inc.

International audience; Nuclear retinoic acid receptor alpha (RARalpha) activates gene expression through dynamic interactions with coregulatory protein complexes, the assembly of which is directed by the ligand and the AF-2 domain of RARalpha. Then RARalpha and its coactivator SRC-3 are degraded by the proteasome. Recently it has emerged that the proteasome also plays a key role in RARalpha-mediated transcription. Here we show that SUG-1, one of the six ATPases of the 19 S regulatory complex of the 26 S proteasome, interacts with SRC-3, is recruited at the promoters of retinoic acid (RA) target genes, and thereby participates to their transcription. In addition, SUG-1 also mediates the proteasomal degradation of SRC-3. However, when present in excess amounts, SUG-1 blocks the activation of RARalpha target genes and the degradation of RARalpha that occurs in response to RA, via its ability to interfere with the recruitment of SRC-3 and other coregulators at the AF-2 domain of RARalpha. We propose a model in which the ratio between SUG-1 and SRC-3 is crucial for the control of RARalpha functioning. This study provides new insights into how SUG-1 has a unique role in linking the transcription and degradation processes via its ability to interact with SRC-3.

International audience; The Morphogenesis and Cell Behaviour meeting held this fall in Barcelona explored the role of forces, adhesion and oscillations in embryonic morphogenesis. It highlighted the impact of new microscopy methods and modelling approaches on the most recent advances of the field.

CD1e displays unique features in comparison with other CD1 proteins. CD1e accumulates in Golgi compartments of immature dendritic cells and is transported directly to lysosomes, where it is cleaved into a soluble form. In these latter compartments, CD1e participates in the processing of glycolipid antigens. In the present study, we show that the N-terminal end of the membrane-associated molecule begins at amino acid 20, whereas the soluble molecule consists of amino acids 32–333. Thus immature CD1e includes an N-terminal propeptide which is cleaved in acidic compartments and so is absent from its mature endosomal form. Mutagenesis experiments demonstrated that the propeptide controls the assembly of the CD1e α-chain with β2-microglobulin, whereas propeptide-deleted CD1e molecules are immunologically active. Comparison of CD1e cDNAs from different mammalian species indicates that the CD1e propeptide is conserved during evolution, suggesting that it may also optimize the generation of CD1e molecules in other species.

International audience; The metabolic impact of the common peroxisome proliferator-activated receptor gamma isoform 2 (PPARgamma2) variant Pro12Ala in human populations has been widely debated. We demonstrate, using a Pro12Ala knockin model, that on chow diet, Ala/Ala mice are leaner, have improved insulin sensitivity and plasma lipid profiles, and have longer lifespans. Gene-environment interactions played a key role as high-fat feeding eliminated the beneficial effects of the Pro12Ala variant on adiposity, plasma lipids, and insulin sensitivity. The underlying molecular mechanisms involve changes in cofactor interaction and adiponectin signaling. Altogether, our results establish the Pro12Ala variant of Ppargamma2 as an important modulator in metabolic control that strongly depends on the metabolic context.

International audience; Notch activity is essential for early T-cell differentiation, but aberrant activity induces T-cell transformation. Thus, Notch target genes must be efficiently silenced in cells where Notch activity is no longer required. How these genes are repressed remains poorly understood. We report here that the Ikaros transcription factor plays a crucial role in repressing the transcriptional response to Notch signaling in T-cell development. Using the Notch target gene Hes-1 as a model, we show that Ikaros and RBP-Jkappa, the transcriptional mediator of Notch signaling, compete for binding to two elements in the Hes-1 promoter in immature thymocytes. This antagonistic interaction likely occurs at the CD4(-) CD8(-) CD3(-) double-negative 4 (DN4) stage, where Ikaros levels and binding to the Hes-1 promoter increase sharply and wild-type thymocytes lose their capacity to transcribe Hes-1 upon Notch stimulation. Nonresponsiveness to Notch signaling requires Ikaros, as Ikaros-deficient DN4 and CD4(+) CD8(+) double-positive (DP) cells remain competent to express Hes-1 after Notch activation. Further, Hes-1 promoter sequences from Ikaros-deficient DP cells show reduced trimethylated H3K27, a modification associated with silent chromatin. These results indicate that Ikaros functions as a transcriptional checkpoint to repress Notch target gene expression in T cells.

International audience; The NAD(+)-dependent deacetylase SIRT1 controls metabolic processes in response to low nutrient availability. We report the metabolic phenotype of mice treated with SRT1720, a specific and potent synthetic activator of SIRT1 that is devoid of direct action on AMPK. SRT1720 administration robustly enhances endurance running performance and strongly protects from diet-induced obesity and insulin resistance by enhancing oxidative metabolism in skeletal muscle, liver, and brown adipose tissue. These metabolic effects of SRT1720 are mediated by the induction of a genetic network controlling fatty acid oxidation through a multifaceted mechanism that involves the direct deacetylation of PGC-1alpha, FOXO1, and p53 and the indirect stimulation of AMPK signaling through a global metabolic adaptation mimicking low energy levels. Combined with our previous work on resveratrol, the current study further validates SIRT1 as a target for the treatment of metabolic disorders and characterizes the mechanisms underlying the therapeutic potential of SIRT1 activation.

International audience; The peripheral nervous system is required for animals to detect and to relay environmental stimuli to central nervous system for the information processing. In Drosophila, the precise spatial and temporal expression of two proneural genes achaete (ac) and scute (sc), is necessary for development of the sensory organs. Here we present an evidence that the transcription co-repressor, dCtBP acts as a negative regulator of sensory organ prepattern. Loss of dCtBP function mutant exhibits ectopic sensory organs, while overexpression of dCtBP results in a dramatic loss of sensory organs. These phenotypes are correlated with mis-emerging of sensory organ precursors and perturbated expression of proneural transcription activator Ac. Mammalian CtBP-1 was identified via interaction with the consensus motif PXDLSX(K/R) of adenovirus E1A oncoprotein. We demonstrated that dCtBP binds directly to PLDLS motif of Drosophila Friend of GATA-1 protein, U-shaped and sharpens the adult sensory organ development. Moreover, we found that dCtBP mediates multivalent interaction with the GATA transcriptional activator Pannier and acts as a direct co-repressor of the Pannier-mediated activation of proneural genes. We demonstrated that Pannier genetically interacts with dCtBP-interacting protein HDAC1, suggesting that the dCtBP-dependent regulation of Pannier activity could utilize a repressive mechanism involving alteration of local chromatine structure.

The progress in sequencing technologies irrigates biology with an ever-increasing number of genome sequences. In most cases, the gene repertoire is predicted in silico and conceptually translated into proteins. As recently highlighted, the predicted genes exhibit frequent errors, particularly in start codons, with a serious impact on subsequent biological studies. A new “ortho-proteogenomic” approach is presented here for the annotation refinement of multiple genomes at once. It combines comparative genomics with an original proteomic protocol that allows the characterization of both N-terminal and internal peptides in a single experiment. This strategy was applied to the Mycobacterium genus with Mycobacterium smegmatis as the reference, and identified 946 distinct proteins, including 443 characterized N termini. These experimental data allowed the correction of 19% of the characterized start codons, the identification of 29 proteins missed during the annotation process, and the curation, thanks to comparative genomics, of 4328 sequences of 16 other Mycobacterium proteomes.

This is an article Open Access.-- et al., Side chain fluorination is often used to make analogs of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] resistant to degradation by 24-hydroxylase. The fluorinated nonsteroidal analogs CD578, WU515, and WY1113 have an increased prodifferentiating action on SW480-ADH colon cancer cells, which correlated with stronger induction of vitamin D receptor (VDR)-coactivator interactions and stronger repression of β-catenin/TCF activity. Cocrystallization of analog CD578 with the zebrafish (z)VDR and an SRC-1 coactivator peptide showed that the fluorine atoms of CD578 make additional contacts with Val444 and Phe448 of activation helix 12 (H12) of the zVDR and with Leu440 of the H11-H12 loop. Consequently, the SRC-1 peptide makes more contacts with the VDR-CD578 complex than with the VDR-1,25(OH)2D3 complex. These data show that fluorination not only affects degradation of an analog but can also have direct effects on H12 stabilization. © 2008 Elsevier Ltd. All rights reserved., This work was supported by grants G.0508.05 and G.0553.06 from Fonds voor Wetenschappelijk Onderzoek (FWO), NucSys (EU Marie Curie RTN), SAF2007-60341 from Ministerio de Educación y Ciencia and RTICC RD06/0020/0009 from Ministerio de Sanidad y Consumo of Spain, SPINE (QLG2-CT-220-0098) and SPINE2-complexes (LSHG-CT-2006-031220) from the European Commission and by CNRS, INSERM, ULP.

International audience; Phosphoinositides are lipid second messengers that are essential for many cellular processes, including signal transduction and cell compartmentalization. Among them, phosphatidylinositol 5-phosphate (PtdIns5P) is the least characterized, although several proteins involved in its regulation are implicated in human diseases. We studied the distribution of 32 PtdIns5P-metabolizing proteins in 39 eukaryotic genomes. Phylogenetic profiles identify four groups of co-evolving proteins, confirming known protein complexes and revealing new ones. The complexes comprise a phosphatase, a kinase and a regulator; this indicates that physical interactions between the three partners are necessary for the acute spatial regulation of PtdIns5P turnover. By examining PtdIns5P metabolism in this new perspective, we propose a role for PtdIns5P in membrane trafficking from late endosomal compartments to the plasma membrane.

International audience; A central issue during embryonic development is to define how different signals cooperate in generating unique cell types. To address this issue, we focused on the function and the regulation of the proneural gene Neurogenin2 (Neurog2) during early mouse spinal neurogenesis. We showed that Neurog2 is first expressed in cells within the neural plate anterior to the node from the 5 somite-stage. The analysis of Neurog2 mutants established a role for this gene in triggering neural differentiation during spinal cord elongation. We identified a 798 base pair enhancer element (Neurog2-798) upstream of the Neurog2 coding sequence that directs the early caudal expression of Neurog2. Embryo culture experiments showed that Retinoic Acid (RA), Sonic hedgehog (Shh) and Fibroblast Growth Factor signals act in concert on this enhancer to control the spatial and temporal induction of Neurog2. We further demonstrated by transgenesis that two RA response elements and a Gli binding site within the Neurog2-798 element are absolutely required for its activity, strongly suggesting that the regulation of Neurog2 early expression by RA and Shh signals is direct. Our data thus support a model where signal integration at the level of a single enhancer constitutes a key mechanism to control the onset of neurogenesis.

International audience; The maturation of cytosolic iron-sulfur (Fe/S) proteins in mammalian cells requires components of the mitochondrial iron-sulfur cluster assembly and export machineries. Little is known about the cytosolic components that may facilitate the assembly process. Here, we identified the cytosolic soluble P-loop NTPase termed huNbp35 (also known as Nubp1) as an Fe/S protein, and we defined its role in the maturation of Fe/S proteins in HeLa cells. Depletion of huNbp35 by RNA interference decreased cell growth considerably, indicating its essential function. The deficiency in huNbp35 was associated with an impaired maturation of the cytosolic Fe/S proteins glutamine phosphoribosylpyrophosphate amidotransferase and iron regulatory protein 1 (IRP1), while mitochondrial Fe/S proteins remained intact. Consequently, huNbp35 is specifically involved in the formation of extramitochondrial Fe/S proteins. The impaired maturation of IRP1 upon huNbp35 depletion had profound consequences for cellular iron metabolism, leading to decreased cellular H-ferritin, increased transferrin receptor levels, and higher transferrin uptake. These properties clearly distinguished huNbp35 from its yeast counterpart Nbp35, which is essential for cytosolic-nuclear Fe/S protein assembly but plays no role in iron regulation. huNbp35 formed a complex with its close homologue huCfd1 (also known as Nubp2) in vivo, suggesting the existence of a heteromeric P-loop NTPase complex that is required for both cytosolic Fe/S protein assembly and cellular iron homeostasis.