New biomarkers for early diagnosis of Lesch-Nyhan disease revealed by metabolic analysis on a large cohort of patients

International audienceBackground: Lesch-Nyhan disease is a rare X-linked neurodevelopemental metabolic disorder caused by a wide variety of mutations in the HPRT1 gene leading to a deficiency of the purine recycling enzyme hypoxanthine-guanine phosphoribosyltransferase (HGprt). The residual HGprt activity correlates with the various phenotypes of Lesch-Nyhan (LN) patients and in particular with the different degree of neurobehavioral disturbances. The prevalence of this disease is considered to be underestimated due to large heterogeneity of its clinical symptoms and the difficulty of diagnosing of the less severe forms of the disease. We therefore searched for metabolic changes that would facilitate an early diagnosis and give potential clues on the disease pathogenesis and potential therapeutic approaches

La vieillesse des uns et le travail des autres

La forte augmentation de la part des « vieux » - en particulier des « très vieux » dans la population totale alimente la croissance des activités de service à la personne et favorise la création en nombre d’emplois salariés caractérisés par une féminisation très élevée et une précarité notoire (importance du temps partiel subi, faible niveau des rémunérations...). Quels sont les savoir-faire et être, non officiellement reconnus en tant que qualifications, mis en œuvre dans le « travail en train de se faire » pour accompagner les plus âgés d’entre nous dans les actes de la vie quotidienne qu’ils ne peuvent plus réaliser seuls ? Que se passe-t-il de significatif au plan sociétal dans les échanges singuliers qui, à cette occasion, se nouent entre des travailleuses salariées réputées peu qualifiées et des retraités âgés ayant besoin d’être aidés ? Plus précisément encore, qu’en est-il des tâches comme « passer du temps à se parler sur la fin de vie et la mort », non officiellement identifiées en tant que telles, mais très récurrentes dans les interventions salariées auprès de personnes âgées en perte d’autonomie, qu’elles soient maintenues à domicile ou placées en institution ? À quelles conditions ces tâches peuvent-elles accéder à la qualification au regard de l’efficacité sociale qu’elles contribuent à produire ? Les différentes contributions composant l’ouvrage s’efforcent de préciser les implications de ces questions et d’y apporter des éléments de réponse. À l’échelle des territoires, la connaissance des modalités et des enjeux du travail des uns dans la vieillesse des autres peut venir en appui aux décisions relatives à la prise en charge des difficultés liées à l’avancée en âge comme aux orientations politiques générales qui la fondent. Dans ses ressorts économiques, sociaux et culturels, cette connaissance donne notamment des repères pour étayer l’alternative actuellement en jeu autour des rapports entre générations dans notre société : d’un côté, poursuivre en l’amplifiant la solidarité salariale via la cotisation sociale ou, de l’autre, encourager la responsabilité individuelle via l’épargne complétée, si nécessaire, par une solidarité/assistance à minima palliant ses défaillances.« Avez-vous remarqué ceci : on s’étonne de devoir mourir bientôt, on s’effraie de ne savoir ni ce que deviendra le monde des vivants dans le futur, ni ce que sera pour nous le monde au-delà, mais s’inquiète-t-on toujours de l’état du monde présent ?... ». Emmanuel Fournier et Jean Christophe Mino, 2008, Les mots des derniers soins, Paris, Les Belles Lettres – extrait d’entretien avec un malade en soins palliatifs – « Ce qui rend malade [au travail], c’est que les gens sont plus grands que les tâches, c’est l’organisation qui est trop petite, irrespectueuse de la qualité du travail possible et des capacités mobilisables par les sujets ». Yves Clot, « C’est le travail qu’il faut soigner ! » in L’Humanité, 20 mai 2008

Metagenomic analysis of Nuclear Quality Control & Degradation mechanisms of Eukaryotic RNA

International audienc

Bacterial Rho helicase: a new tool to dissect mRNP biogenesis and quality control in yeast

International audienceIn eukaryotic cells, the co-transcriptional mRNA processing and packaging reactions that lead to the formation of export competent messenger ribonucleoprotein particles (mRNPs) are under the surveillance of quality control (QC) steps. Aberrant mRNPs resulting from faulty events are detected by the QC apparatus and retained in the nucleus with ensuing elimination of their mRNA component by the RNA degradation machinery. A decade of biochemical and genetic experiments in yeast allowed the identification of the nuclear degradation machinery including the core exosome and its two associated catalytic subunits Rrp6p and Rrp44p, its cofactors Rrp47p and Mpp6p as well as the activator complex TRAMP. Similarly, studies of the THO-Sub2 complex of the mRNP assembly and export apparatus have provided valuable information on the nuclear retention and degradation of a particular class of aberrant mRNPs. However, a unifying mechanism of action underlying the QC process remains elusive. Here, we review the implementation of a new experimental approach whereby the production of aberrant mRNPs is massively increased upon heterologous expression of the bacterial Rho helicase in yeast. Using this methodology, we have shown that the QC process is coordinated by Nrd1p (a component of the early termination complex) whose increased co-transcriptional recruitment promotes the attachment of the 3’-5’ exonuclease Rrp6p along with the co-factors Rrp47p and Mpp6p. Interestingly, we established that Rrp6p functions independently from the core exosome, yet is stimulated by two forms of the TRAMP complex that include Trf4p or Trf5p and Air2p but not Air1p. The results suggest that specific substrates could be primed for decay via various QC pathways owing to the versatility of the mRNA degradation apparatus. In this context, the bacterial Rho helicase provides a valuable tool to decipher the QC molecular process in yeast and possibly the homologous process in mammalian cells

Analyse métagénomique du mécanisme de discrimination entre les transcrits codants et non-codants par la machinerie de dégradation de l’ARN

National audienc

Yeast RNA exosome activity is necessary for maintaining cell wall stability through proper protein glycosylation

International audienceNuclear RNA exosome is the main 3′→5′ RNA degradation and processing complex in eukaryotic cells and its dysregulation therefore impacts gene expression and viability. In this work we show that RNA exosome activity is necessary for maintaining cell wall stability in yeast Saccharomyces cerevisiae. While the essential RNA exosome catalytic subunit Dis3 provides exoribonuclease catalytic activity, the second catalytic subunit Rrp6 has a noncatalytic role in this process. RNA exosome cofactors Rrp47 and Air1/2 are also involved. RNA exosome mutants undergo osmoremedial cell lysis at high temperature or at physiological temperature upon treatment with cell wall stressors. Finally, we show that a defect in protein glycosylation is a major reason for cell wall instability of RNA exosome mutants. Genes encoding enzymes that act in the early steps of the protein glycosylation pathway are down-regulated at high temperature in cells lacking Rrp6 protein or Dis3 exoribonuclease activity and overexpression of the essential enzyme Psa1, that catalyzes synthesis of the mannosylation precursor, suppresses temperature sensitivity and aberrant morphology of these cells. Furthermore, this defect is connected to a temperature-dependent increase in accumulation of noncoding RNAs transcribed from loci of relevant glycosylation-related genes

Yeast RNA exosome activity is necessary for maintaining cell wall stability through proper protein glycosylation

International audienceNuclear RNA exosome is the main 3′→5′ RNA degradation and processing complex in eukaryotic cells and its dysregulation therefore impacts gene expression and viability. In this work we show that RNA exosome activity is necessary for maintaining cell wall stability in yeast Saccharomyces cerevisiae. While the essential RNA exosome catalytic subunit Dis3 provides exoribonuclease catalytic activity, the second catalytic subunit Rrp6 has a noncatalytic role in this process. RNA exosome cofactors Rrp47 and Air1/2 are also involved. RNA exosome mutants undergo osmoremedial cell lysis at high temperature or at physiological temperature upon treatment with cell wall stressors. Finally, we show that a defect in protein glycosylation is a major reason for cell wall instability of RNA exosome mutants. Genes encoding enzymes that act in the early steps of the protein glycosylation pathway are down-regulated at high temperature in cells lacking Rrp6 protein or Dis3 exoribonuclease activity and overexpression of the essential enzyme Psa1, that catalyzes synthesis of the mannosylation precursor, suppresses temperature sensitivity and aberrant morphology of these cells. Furthermore, this defect is connected to a temperature-dependent increase in accumulation of noncoding RNAs transcribed from loci of relevant glycosylation-related genes

Perturbation of mRNP biogenesis reveals a dynamic landscape of the Rrp6-dependent surveillance machinery trafficking along the yeast genome

<p>Eukaryotic cells have evolved a nuclear quality control (QC) system to monitor the co-transcriptional mRNA processing and packaging reactions that lead to the formation of export-competent ribonucleoprotein particles (mRNPs). Aberrant mRNPs that fail to pass the QC steps are retained in the nucleus and eliminated by the exonuclease activity of Rrp6. It is still unclear how the surveillance system is precisely coordinated both physically and functionally with the transcription machinery to detect the faulty events that may arise at each step of transcript elongation and mRNP formation. To dissect the QC mechanism, we previously implemented a powerful assay based on global perturbation of mRNP biogenesis in yeast by the bacterial Rho helicase. By monitoring model genes, we have shown that the QC process is coordinated by Nrd1, a component of the NNS complex (Nrd1-Nab3-Sen1) involved in termination, processing and decay of ncRNAs which is recruited by the CTD of RNAP II. Here, we have extended our investigations by analyzing the QC behaviour over the whole yeast genome. We performed high-throughput RNA sequencing (RNA-seq) to survey a large collection of mRNPs whose biogenesis is affected by Rho action and which can be rescued upon Rrp6 depletion. This genome-wide perspective was extended by generating high-resolution binding landscapes (ChIP-seq) of QC components along the yeast chromosomes before and after perturbation of mRNP biogenesis. Our results show that perturbation of mRNP biogenesis redistributes the QC components over the genome with a significant hijacking of Nrd1 and Nab3 from genomic loci producing ncRNAs to Rho-affected protein-coding genes, triggering termination and processing defects of ncRNAs.</p

Tho2 is critical for the recruitment of Rrp6 to chromatin in response to perturbed mRNP biogenesis

International audienceThe eukaryotic THO complex coordinates the assembly of so-called messenger RNA-ribonucleoprotein particles (mRNPs), a process that involves co-transcriptional coating nascent mRNAs with proteins. Once formed, mRNPs undergo quality control step marks them either for active transport to cytoplasm, or Rrp6/RNA exosome-mediated degradation in nucleus. However, mechanism behind is still unclear. We investigated budding yeast by expressing bacterial Rho helicase, which globally perturbs mRNP formation. examined genome-wide binding profiles subunits Tho2, Thp2, Hpr1, and Mft1 upon perturbation biogenesis, found Tho2 plays two roles. In addition its function as subunit complex, biogenesis targets Rrp6 chromatin via C-terminal domain. Interestingly, other are not enriched on necessary localizing at target loci. Our study highlights potential role control, independent subunits. Considering both RNA exosome evolutionarily highly conserved, our findings likely relevant surveillance mammals