The DEAD-box helicase Ded1 from yeast is an mRNP cap-associated protein that shuttles between the cytoplasm and nucleus

International audienceThe DEAD-box helicase Ded1 is an essential yeast protein that is closely related to mammalian DDX3 and to other DEAD-box proteins involved in developmental and cell cycle regulation. Ded1 is considered to be a translation-initiation factor that helps the 40S ribosome scan the mRNA from the 5 7-methylguanosine cap to the AUG start codon. We used IgG pull-down experiments, mass spectrom-etry analyses, genetic experiments, sucrose gradients , in situ localizations and enzymatic assays to show that Ded1 is a cap-associated protein that actively shuttles between the cytoplasm and the nucleus. NanoLC-MS/MS analyses of purified complexes show that Ded1 is present in both nuclear and cytoplasmic mRNPs. Ded1 physically interacts with purified components of the nuclear CBC and the cytoplasmic eIF4F complexes, and its enzymatic activity is stimulated by these factors. In addition, we show that Ded1 is genetically linked to these factors. Ded1 comigrates with these proteins on sucrose gradients, but treatment with rapamycin does not appreciably alter the distribution of Ded1; thus, most of the Ded1 is in stable mRNP complexes. We conclude that Ded1 is an mRNP cofactor of the cap complex that may function to remodel the different mRNPs and thereby regulate the expression of the mRNAs

Xanthomonas arboricola pv. juglandis and pv. corylina: Brothers or distant relatives? : genetic clues, epidemiology, and insights for disease management

Background: The species Xanthomonas arboricola comprises up to nine pathovars, two of which affect nut crops: pv. juglandis, the causal agent of walnut bacterial blight, brown apical necrosis, and the vertical oozing canker of Persian (English) walnut; and pv. corylina, the causal agent of the bacterial blight of hazelnut. Both pathovars share a complex population structure, represented by different clusters and several clades. Here we describe our current understanding of symptomatology, population dynamics, epidemiology, and disease control. Taxonomic status: Bacteria; Phylum Proteobacteria; Class Gammaproteobacteria; Order Lysobacterales (earlier synonym of Xanthomonadales); Family Lysobacteraceae (earlier synonym of Xanthomonadaceae); Genus Xanthomonas; Species X. arboricola; Pathovars: pv. juglandis and pv. corylina. Host range and symptoms: The host range of each pathovar is not limited to a single species, but each infects mainly one plant species: Juglans regia (X. arboricola pv. juglandis) and Corylus avellana (X. arboricola. pv. corylina). Walnut bacterial blight is characterized by lesions on leaves and fruits, and cankers on twigs, branches, and trunks; brown apical necrosis symptoms consist of apical necrosis originating at the stigmatic end of the fruit. A peculiar symptom, the vertical oozing canker developing along the trunk, is elicited by a particular genetic lineage of the bacterium. Symptoms of hazelnut bacterial blight are visible on leaves and fruits as necrotic lesions, and on woody parts as cankers. A remarkable difference is that affected walnuts drop abundantly, whereas hazelnuts with symptoms do not. Distribution: Bacterial blight of walnut has a worldwide distribution, wherever Persian (English) walnut is cultivated; the bacterial blight of hazelnut has a more limited distribution, although disease outbreaks are currently more frequently reported. X. arboricola pv. juglandis is regulated almost nowhere, whereas X. arboricola pv. corylina is regulated in most European and Mediterranean Plant Protection Organization (EPPO) countries. Epidemiology and control: For both pathogens infected nursery material is the main pathway for their introduction and spread into newly cultivated areas; additionally, infected nursery material is the source of primary inoculum. X. arboricola pv. juglandis is also disseminated through pollen. Disease control is achieved through the phytosanitary certification of nursery material (hazelnut), although approved certification schemes are not currently available. Once the disease is present in walnut/hazelnut groves, copper compounds are widely used, mostly in association with dithiocarbamates; where allowed, antibiotics (preferably kasugamycin) are sprayed. The emergence of strains highly resistant to copper currently represents the major threat for effective management of the bacterial blight of walnut

Mise en évidence de protéines révélant une réponse au stress adaptative divergente entre les espèces Dreissena polymorpha et Dreissena rostriformis bugensis

International audienceZebra mussels Dreissena polymorpha, are bivalve molluscs used in ecotoxicology, as a sentinel species with, among other things, a strong bioaccumulation capacity. The quagga mussel, Dreissena rostriformis bugensis, has more recently colonized Western Europe. Also invasive, it competes with zebra mussels for habitats and, in some areas, has completely replaced it. Its use as a sentinel species is envisaged but requires understanding the mechanisms involved during the adaptive stress response and comparing them to those of the zebra mussel, which are better characterized. With this in mind, an exposure of the two species to a classical contaminant was performed to compare their responses. The individuals were exposed to a concentration of 100 μg / L of cadmium for 7 days, and then the gill proteins were separated by two-dimensional electrophoresis and the variable abundance proteoforms were identified by mass spectrometry. The functional analysis reveals promising elements for the study of the adaptive response in both species. A difference of expression of actors of the energy metabolism pathways points a physiological difference, with a reallocation of energy as well as the appearance of truncated proteins.Les moules zébrées, Dreissena polymorpha sont des mollusques bivalves utilisés en écotoxicologie comme espèces sentinelles du fait, entre autre, de leur capacité de bioaccumulation. La moule quagga, Dreissena rostriformis bugensis, a colonisé plus récemment l'Europe occidentale. Egalement invasive, elle est en compétition avec la moule zébrée pour les habitats et, dans certaines zones, l'a complètement remplacée. Son utilisation en tant qu'espèce sentinelle est envisagée mais nécessite de comprendre les mécanismes mis en jeu au cours de la réponse adaptative au stress et de les comparer à ceux de la moule zébrée, mieux caractérisés. Dans cette optique, une exposition des deux espèces à un contaminant classique a été réalisée afin de comparer leurs réponses. Les individus ont été exposés à une concentration de 100μg/L de cadmium pendant 7 jours, puis les protéines des branchies ont été séparées par électrophorèse bidimensionnelle et les protéoformes d'abondance variable ont été identifiées par spectrométrie de masse. L'analyse fonctionnelle révèle des éléments prometteurs pour l'étude de la réponse adaptative chez les deux espèces. Une différence d'expression d'acteurs des voies du métabolisme énergétique pointe une différence physiologique, avec une réallocation de l'énergie ainsi que l'apparition de protéines tronquées

A Conserved Noncoding Locus Regulates Random Monoallelic Xist Expression across a Topological Boundary

cis-Regulatory communication is crucial in mammalian development and is thought to be restricted by the spatial partitioning of the genome in topologically associating domains (TADs). Here, we discovered that the Xist locus is regulated by sequences in the neighboring TAD. In particular, the promoter of the noncoding RNA Linx (LinxP) acts as a long-range silencer and influences the choice of X chromosome to be inactivated. This is independent of Linx transcription and independent of any effect on Tsix, the antisense regulator of Xist that shares the same TAD as Linx. Unlike Tsix, LinxP is well conserved across mammals, suggesting an ancestral mechanism for random monoallelic Xist regulation. When introduced in the same TAD as Xist, LinxP switches from a silencer to an enhancer. Our study uncovers an unsuspected regulatory axis for X chromosome inactivation and a class of cis-regulatory effects that may exploit TAD partitioning to modulate developmental decisions.Galupa et al. uncover elements important for Xist regulation in its neighboring TAD and reveal that these elements can influence gene regulation both within and between topological domains. These findings, in a context where dynamic, developmental expression is necessary, challenge current models for TAD-based gene-regulatory landscapes

Correlation of fluorescence evolution for quantitative analysis of labels and sensors

International audienceTitration without separation, e.g. quantification of a target species in living cells, is a challenge of analytical chemistry. We perform the selective detection of a target using the kinetics involved in a photochemical process and develop a correlation method that we illustrate by the titration of a fluorescent photoswitcher and the target of a photoswitching sensor. Correlating an input time series and a well-chosen weighting function associated with a variable characteristic time yields a spectrum of characteristic times. The upper integration limit of the correlation output can be chosen to match the argument of an extremum of the spectrum with a characteristic time of the input time series in order to quantify the target. A similar procedure is followed to optimize the signal-to-noise ratio. Selectivity and signal-tonoise ratio associated with 15 weighting functions are theoretically predicted. The results are applied to the titration of the reversibly photoswitchable fluorescent protein Dronpa-2 and the titration of calcium using a reversibly photoswitchable fluorescent sensor. The performance of the correlation method is favorably compared to the one of other dynamic contrast protocols

Dynamic contrast with reversibly photoswitchable fluorescent labels for imaging living cells

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Dynamic contrast for overcoming spectral interferences in fluorescence imaging

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