1,721,012 research outputs found
Composition and dynamic of Nonsense-mediated mRNA decay complexes in yeast
No full textLe Nonsense-Mediated mRNA Decay (NMD) détecte et dégrade les ARNs qui ont une terminaison de la traduction précoce. Il affecte une grande diversité d’ARNs cytoplasmiques, c’est la voie majeure de dégradation des ARNs aberrants.Les ARNs ciblés par le NMD chez la levure ont une phase ouverte de lecture courte et un long 3’-UTR. Comment ces caractéristiques permettent une dégradation efficace par le NMD et quelles sont les étapes du mécanisme reste peu clair. À partir de 112 purifications d’affinités suivies d’une analyse en spectrométrie de masse quantitative, nous avons identifié deux complexes distincts impliqués dans le NMD. Ces deux complexes, mutuellement exclusifs, s’articulent autour d’UPF1, la protéine majeure du NMD. Le premier complexe, nommé Détecteur, aurait un rôle dans la reconnaissance des cibles alors que le deuxième complexe, nommé Effecteur, initierait la dégradation des ARNs par une interaction directe avec la machinerie de décapping.Les facteurs impliqués dans ce modèle sont tous conservés à travers les eucaryotes et les étapes décrites sont transposables d’après la littérature. Cela semble indiquer un nouveau paradigme pour le mécanisme du NMD qui s’articulerait autour d’une base universelle commune à laquelle pourrait s’ajouter des étapes spécifiques des organismes ou des types d’ARNs.Nonsense-Mediated mRNA Decay (NMD) detects and degrades RNA for which translation ends prematurely. It affects a large diversity of cytoplasmics RNAs; it is the major decay pathway for aberrants RNAs.In yeast, NMD targeted RNAs have a short open reading frame and a long 3’-UTR. How these two features lead to an efficient degradation through NMD and what are the steps of this mechanism is still unclear.From 112 affinity purifications of NMD factors followed by an analysis using quantitative mass spectrometry, we identified two distinct complexes. Those complexes were mutually exclusive and both contained Upf1, the major NMD protein. A first complex, named Detector, might have a role in the NMD substrates recognition whereas the second one, named Effector, would initiate the degradation through a direct interaction with the decapping machinery.The factors involved in our new model are all conserved throughout eukaryotes and the steps we describe have potential equivalents in other species. Our data suggest a new paradigm for the NMD mechanism that would be organised around a shared universal base to which specific steps could be added in certain organisms or for certain types of RNA substrates
Mechanisms of RNA degradation, dependent or independent of deadenylation
No full textLa dégradation de l'ARN est une étape essentielle pour maintenir l'équilibre nécessaire à l'expression des gènes et à leur régulation. Chez les eucaryotes, le modèle le plus courant propose que la dégradation de l'ARN est contrôlée par la vitesse de raccourcissement de la queue poly-A. En dessous d'un certain seuil, la présence d'une courte queue oligo-A entraîne l'activation du décapping et la dégradation de l'ARN. Cependant, la déadénylation progressive n'est pas nécessaire pour une voie majeure de dégradation rapide de l'ARN, la dégradation par le "nonsense-mediated mRNA decay" (NMD), qui se passe indépendamment de la taille d'une queue poly-A. Sur la base de mesures à grande échelle de la longueur de la queue poly-A et d'estimations de la demi-vie de l'ARN, nous proposons qu'un modèle de dégradation indépendant de la déadénylation, similaire au NMD, puisse exister pour d'autres ARN instables. Ce modèle prédit que des changements de vitesse de déadénylation ne devraient pas avoir d'impact sur la demi-vie de ces ARN. Le raccourcissement de la queue des poly-A semble être dans ce cas un processus parallèle et non pas nécessaire pour la dégradation de l'ARN. Comme prédit par notre modèle, un ARN rapporteur instable et insensible au NMD n'est pas stabilisé par une diminution de la vitesse de déadénylation, même s'il est très sensible à l'inactivation du décapping. Ces résultats ont été obtenus dans un système "degron" de déplétion rapide et spécifique de protéines d’intérêt, qui permet d’éviter une perturbation globale du métabolisme des cellules testées. En accord avec ces résultats fonctionnels, nous avons identifié les ARN instables comme une population de molécules d'ARN associées à la protéine de liaison cytoplasmique des poly-A et conclu que de longues queues de poly-A sont une caractéristique naturelle des ARN instables. Nos résultats sont compatibles avec les estimations récentes de la taille des queues poly-A et de la vitesse de dégradation des transcrits et devraient avoir un impact important sur la découverte des mécanismes moléculaires par lesquels la dégradation des ARN est initiée chez les eucaryotes.RNA degradation is a critical step in maintaining the proper balance required for gene expression and its regulation. In eukaryotes, RNA decay is thought to be controlled by the speed of poly-A tail shortening. Below a threshold, the presence of an oligo-A tail leads to decapping activation and rapid RNA degradation. However, progressive deadenylation is not required for a major fast RNA degradation pathway, the nonsense mediated mRNA decay (NMD), which occurs independently of the size of a poly-A tail. Based on large-scale poly-A tail length measurements and RNA half-life estimates, we hypothesize that a deadenylation-independent degradation model, similar to NMD, applies to other unstable RNAs. This model predicts that changes in the deadenylation speed should not impact the half-life of these RNAs. Poly-A tail shortening could be thus considered as a parallel event rather than a requirement for RNA degradation. We found that, as predicted by a deadenylation-independent model of RNA degradation, an unstable reporter RNA that is insensitive to NMD, is not stabilized by an inhibition of deadenylation, even if it is highly sensitive to the decapping activity. These results were obtained in a "degron" system for quick and specific depletion of the proteins, which allows a minimal global perturbation of the tested cells. In line with these functional results, we identified unstable RNAs as a population of RNA molecules associated with the poly-A binding protein and conclude that long poly-A tails are a natural feature of unstable RNAs. Our results are consistent with recent large-scale estimates of poly-A tails length and RNA stability and imply that molecular mechanisms involved in the initiation of RNA degradation in eukaryotes remain to be discovered
60S ribosomal subunit assembly dynamics defined by semi-quantitative mass spectrometry of purified complexes
No full textDuring the highly conserved process of eukaryotic ribosome formation, RNA follows a maturation path with well-defined, successive intermediates that dynamically associate with many pre-ribosomal proteins. A comprehensive description of the assembly process is still lacking. To obtain data on the timing and order of association of the different pre-ribosomal factors, a strategy consists in the use of pre-ribsomal particles isolated from mutants that block ribosome formation at different steps. Immunoblots, inherently limited to only a few factors, have been applied to evaluate the accumulation or decrease of pre-ribosomal intermediates under mutant conditions. For a global protein-level description of different 60S ribosomal subunit maturation intermediates in yeast, we have adapted a method of in vivo isotopic labelling and mass spectrometry to study pre-60S complexes isolated from strains in which rRNA processing was affected by individual depletion of five factors: Ebp2, Nog1, Nsa2, Nog2 or Pop3. We obtained quantitative data for 45 distinct pre-60S proteins and detected coordinated changes for over 30 pre-60S factors in the analysed mutants. These results led to the characterisation of the composition of early, intermediate and late pre-ribosomal complexes, specific for crucial maturation steps during 60S assembly in eukaryotes
Toxicity of a novel antifungal compound is modulated by endoplasmic reticulum-associated protein degradation components
No full textIn a search for new antifungal compounds, we screened a library of 4454 chemicals for toxicity against the human fungal pathogen Aspergillus fumigatus. We identified sr7575, a molecule that inhibits growth of the evolutionary distant fungi A. fumigatus, Cryptococcus neoformans, Candida albicans, and Saccharomyces cerevisiae but lacks acute toxicity for mammalian cells. To gain insight into the mode of inhibition, sr7575 was screened against 4885 S. cerevisiae mutants from the systematic collection of haploid deletion strains and 977 barcoded haploid DAmP strains in which the function of essential genes was perturbed by the introduction of a drug resistance cassette downstream of the coding sequence region. Comparisons with previously published chemogenomic screens revealed that the set of mutants conferring sensitivity to sr7575 was strikingly narrow, affecting components of the endoplasmic-associated protein degradation (ERAD) stress response and the ER membrane protein complex (EMC). ERAD-deficient mutants were hypersensitive to sr7575 in both S. cerevisiae and A. fumigatus, indicating a conserved mechanism of growth inhibition between yeast and filamentous fungi. Although the unfolded protein response (UPR) is linked to ERAD regulation, sr7575 did not trigger the UPR in A. fumigatus and UPR mutants showed no enhanced sensitivity to the compound. The data from this chemogenomic analysis demonstrate that sr7575 exerts its antifungal activity by disrupting ER protein quality control in a manner that requires ERAD intervention but bypasses the need for the canonical UPR. ER protein quality control is thus a specific vulnerability of fungal organisms that might be exploited for antifungal drug development
Minimal perturbation analysis of mRNA degradation rates with Tet-off and RT-qPCR
No full textMessenger RNA stability is an important variable in gene expression and its dynamics. High stability ensures a constant level of synthesized protein, whereas mRNA instability can be critical for regulatory processes in which protein production needs to be stopped, such as development, inflammation or adaptation to stress. Accurate measurements of RNA degradation rates are important for understanding how RNA features and RNA binding proteins affect the post-transcriptional life of an mRNA. As an alternative to global transcriptional inhibition methods, the use of a Tet-off repressible promoter has the advantage that cells are minimally perturbed by the addition of doxycyclin during the assay. We illustrate the use of a reporter mRNA expressed from a plasmid in Saccharomyces cerevisiae cells, but similar methods can be applied to other regulated promoters, on plasmids or by genome editing, and in other organisms. RNA levels are measured by reverse transcription followed by quantitative PCR. An exponential decay law is then used to estimate how well the measurements follow this expected trend for the simplest possible mechanism of RNA degradation, where the decay is proportional to the amount of RNA present at any given time
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Composition and Dynamics of Protein Complexes Measured by Quantitative Mass Spectrometry of Affinity-Purified Samples
No full textInternational audienceMultiple protein complexes are fundamental parts of living systems. Identification of the components of these complexes and characterization of the molecular mechanisms that allow their formation, function and regulation can be done by affinity purification of proteins and associated factors followed by mass spectrometry of peptides. Speed and specificity for the isolation of complexes from whole cell extracts improved over time together with the reliable identification and quantification of proteins by mass spectrometry. Relative quantification of proteins in such samples can now be done to characterize even relatively non-abundant complexes. We describe here our experience with proteins fused with the Z domain, derived from staphylococcal protein A, and IgG affinity purification for the analysis of protein complexes involved in RNA metabolism in the budding yeast Saccharomyces cerevisiae. We illustrate the use of enrichment calculations for proteins in purified samples as a way to robust identification of protein partners. While the protocols presented here are specific for yeast, their principles can be applied to the study of protein complexes in any other organism
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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