Sumoylation of the THO complex regulates the biogenesis of a subset of mRNPs

International audienceAssembly of messenger ribonucleoparticles (mRNPs) is a pivotal step in gene expression, but only a few molecular mechanisms contributing to its regulation have been described. Here, through a comprehensive proteomic survey of mRNP assembly, we demonstrate that the SUMO pathway specifically controls the association of the THO complex with mRNPs. We further show that the THO complex, a key player in the interplay between gene expression, mRNA export and genetic stability, is sumoylated on its Hpr1 subunit and that this modification regulates its association with mRNPs. Altered recruitment of the THO complex onto mRNPs in sumoylation-defective mutants does not affect bulk mRNA export or genetic stability, but impairs the expression of acidic stress-induced genes and, consistently, compromises viability in acidic stress conditions. Importantly, inactivation of the nuclear exosome suppresses the phenotypes of the hpr1 non-sumoylatable mutant, showing that SUMO-dependent mRNP assembly is critical to allow a specific subset of mRNPs to escape degradation. This article thus provides the first example of a SUMO-dependent mRNP-assembly event allowing a refined tuning of gene expression, in particular under specific stress conditions

Study of the regulation of messenger RNA metabolism in the yeast Saccharomyces cerevisiae

Au cours de la transcription, plusieurs facteurs sont assemblés sur les ARN messagers pour former des Ribonucléoparticules de messagers (mRNPs), et contrôler leur maturation, leur stabilité et leur devenir dans le cytoplasme. Afin d’assurer la production de protéines fonctionnelles, la cellule dispose de plusieurs mécanismes de régulation et de contrôle de qualité assurant la fidélité de l’information génétique transmise au niveau ARN messager et protéine.Chez la levure Saccharomyces cerevisiae, un ensemble de protéines associées au pore nucléaire, incluant la SUMO protéase Ulp1, a été impliqué dans un contrôle de qualité des mRNPs régulant leur export vers le cytoplasme. Ces données suggéraient que l’export des ARN messagers pourrait être contrôlé par la modification post-traductionnelle par le polypeptide SUMO d’un ou de plusieurs effecteurs au sein des mRNPs. Afin de mieux comprendre ces processus, nous avons combiné plusieurs approches visant à identifier ces protéines SUMOylées. En particulier, nous avons mis en place un crible protéomique visant à identifier les protéines dont l’association sur les mRNPs dépend d’Ulp1. Ce crible nous a permis de mettre en évidence une régulation par Ulp1 de l’assemblage du complexe THO sur les ARN messagers. Ce complexe, recruté sur les gènes et les mRNPs, est connu pour contribuer à l’efficacité de la transcription, prévenir l’instabilité génétique liée à la formation d’hybrides ADN matrice – ARN messager (dénommés R-loops) et permettre l’export des mRNPs. En combinant l’analyse biochimique de différentes catégories de mRNPs à des expériences d’immunoprécipitation de l’ARN, nous avons montré que l’activité de la SUMO-protéase Ulp1 est nécessaire à l’association du complexe THO sur différents ARN messagers. De plus, nous avons montré que le complexe THO est SUMOylé sur le domaine C-terminal de sa sous-unité Hpr1, et que Ulp1 régule cette modification. Enfin, cet événement de SUMOylation du complexe THO régule son association avec les mRNPs. L’analyse fonctionnelle de mutants affectant la SUMOylation du complexe THO révèle que des défauts de SUMOylation de ce complexe compromettent ses fonctions dans la transcription sans affecter l’export. De manière intéressante, nous avons observé que la présence d’un intron sur des rapporteurs LacZ diminue la sensibilité de leur expression à des inactivations ou des défauts de SUMOylation du complexe THO. Ce phénotype entraine une augmentation relative des niveaux d’ARN pré-messagers dans ces mutants, un phénomène rendant compte de la fuite cytoplasmique apparente d’ARN non épissés précédemment observée dans le mutant ulp1. L’ensemble de ces données caractérise pour la première fois un rôle de la SUMOylation dans le contrôle de l’assemblage et du devenir cellulaire des mRNPs.During transcription, several factors associate with mRNA to form messenger Ribonucleoparticles (mRNPs), thereby controlling their processing, their stability, and their cytoplasmic fate. To ensure the production of functional proteins from these mRNAs, eukaryotic cells contain numerous regulatory and quality control systems in order to prevent aberrant mRNP accumulation and export.In the yeast Saccharomyces cerevisiae, several nuclear pore associated proteins, including the SUMO isopeptidase Ulp1, have been involved in a mRNP quality control regulating their nuclear export. These data suggested that post-translational modification by SUMO of one or several mRNP components could regulate mRNA export. In order to understand the molecular mechanisms underlying this process, we undertook several approaches to identify these SUMOylated factors. In particular, we have set up a proteomic screen to identify mRNP components whose assembly onto mRNPs depends on Ulp1 activity.This proteomic survey revealed an Ulp1-dependent regulation of THO complex assembly to mRNPs. This complex, recruited to transcribed genes and mRNPs, is known to regulate transcription elongation by preventing DNA-RNA hybrids formation (termed R-loops), and mRNP export. Through a combination of proteomic analysis of mRNPs assembled in Ulp1 mutant cells, with RNA / chromatin immunoprecipitation experiments, we demonstrate that Ulp1 controls specifically the recruitment of the THO complex within mRNPs. SUMOylation analysis further reveals that Ulp1 targets the THO complex subunit Hpr1 on its C-terminal domain for deSUMOylation. We further show that this SUMOylation event regulates THO complex association within mRNPs. Finally, functional analysis reveal that impaired deSUMOylation of the THO complex do not affect mRNP export, but disturbs expression of LacZ reporter genes, a phenotype classically associated with THO complex dysfunction. Intriguingly, the transcriptional effect of inactivation or impaired deSUMOylation of the THO complex on LacZ expression is alleviated by the presence of an intron, providing a molecular basis for previously reported pre-mRNA leakage phenotypes. Our data therefore unravels for the first time a function of SUMO in the control of mRNP assembly contributing to proper mRNP homeostasis

Etude de la régulation du métabolisme des ARN messagers chez la levure Saccharomyces cerevisiae

Au cours de la transcription, plusieurs facteurs sont assemblés sur les ARN messagers pour former des Ribonucléoparticules de messagers (mRNPs), et contrôler leur maturation, leur stabilité et leur devenir dans le cytoplasme. Afin d assurer la production de protéines fonctionnelles, la cellule dispose de plusieurs mécanismes de régulation et de contrôle de qualité assurant la fidélité de l information génétique transmise au niveau ARN messager et protéine.Chez la levure Saccharomyces cerevisiae, un ensemble de protéines associées au pore nucléaire, incluant la SUMO protéase Ulp1, a été impliqué dans un contrôle de qualité des mRNPs régulant leur export vers le cytoplasme. Ces données suggéraient que l export des ARN messagers pourrait être contrôlé par la modification post-traductionnelle par le polypeptide SUMO d un ou de plusieurs effecteurs au sein des mRNPs. Afin de mieux comprendre ces processus, nous avons combiné plusieurs approches visant à identifier ces protéines SUMOylées. En particulier, nous avons mis en place un crible protéomique visant à identifier les protéines dont l association sur les mRNPs dépend d Ulp1. Ce crible nous a permis de mettre en évidence une régulation par Ulp1 de l assemblage du complexe THO sur les ARN messagers. Ce complexe, recruté sur les gènes et les mRNPs, est connu pour contribuer à l efficacité de la transcription, prévenir l instabilité génétique liée à la formation d hybrides ADN matrice ARN messager (dénommés R-loops) et permettre l export des mRNPs. En combinant l analyse biochimique de différentes catégories de mRNPs à des expériences d immunoprécipitation de l ARN, nous avons montré que l activité de la SUMO-protéase Ulp1 est nécessaire à l association du complexe THO sur différents ARN messagers. De plus, nous avons montré que le complexe THO est SUMOylé sur le domaine C-terminal de sa sous-unité Hpr1, et que Ulp1 régule cette modification. Enfin, cet événement de SUMOylation du complexe THO régule son association avec les mRNPs. L analyse fonctionnelle de mutants affectant la SUMOylation du complexe THO révèle que des défauts de SUMOylation de ce complexe compromettent ses fonctions dans la transcription sans affecter l export. De manière intéressante, nous avons observé que la présence d un intron sur des rapporteurs LacZ diminue la sensibilité de leur expression à des inactivations ou des défauts de SUMOylation du complexe THO. Ce phénotype entraine une augmentation relative des niveaux d ARN pré-messagers dans ces mutants, un phénomène rendant compte de la fuite cytoplasmique apparente d ARN non épissés précédemment observée dans le mutant ulp1. L ensemble de ces données caractérise pour la première fois un rôle de la SUMOylation dans le contrôle de l assemblage et du devenir cellulaire des mRNPs.During transcription, several factors associate with mRNA to form messenger Ribonucleoparticles (mRNPs), thereby controlling their processing, their stability, and their cytoplasmic fate. To ensure the production of functional proteins from these mRNAs, eukaryotic cells contain numerous regulatory and quality control systems in order to prevent aberrant mRNP accumulation and export.In the yeast Saccharomyces cerevisiae, several nuclear pore associated proteins, including the SUMO isopeptidase Ulp1, have been involved in a mRNP quality control regulating their nuclear export. These data suggested that post-translational modification by SUMO of one or several mRNP components could regulate mRNA export. In order to understand the molecular mechanisms underlying this process, we undertook several approaches to identify these SUMOylated factors. In particular, we have set up a proteomic screen to identify mRNP components whose assembly onto mRNPs depends on Ulp1 activity.This proteomic survey revealed an Ulp1-dependent regulation of THO complex assembly to mRNPs. This complex, recruited to transcribed genes and mRNPs, is known to regulate transcription elongation by preventing DNA-RNA hybrids formation (termed R-loops), and mRNP export. Through a combination of proteomic analysis of mRNPs assembled in Ulp1 mutant cells, with RNA / chromatin immunoprecipitation experiments, we demonstrate that Ulp1 controls specifically the recruitment of the THO complex within mRNPs. SUMOylation analysis further reveals that Ulp1 targets the THO complex subunit Hpr1 on its C-terminal domain for deSUMOylation. We further show that this SUMOylation event regulates THO complex association within mRNPs. Finally, functional analysis reveal that impaired deSUMOylation of the THO complex do not affect mRNP export, but disturbs expression of LacZ reporter genes, a phenotype classically associated with THO complex dysfunction. Intriguingly, the transcriptional effect of inactivation or impaired deSUMOylation of the THO complex on LacZ expression is alleviated by the presence of an intron, providing a molecular basis for previously reported pre-mRNA leakage phenotypes. Our data therefore unravels for the first time a function of SUMO in the control of mRNP assembly contributing to proper mRNP homeostasis.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Nuclear pore components affect distinct stages of intron-containing gene expression.

International audienceSeveral nuclear pore-associated factors, including the SUMO-protease Ulp1, have been proposed to prevent the export of intron-containing messenger ribonucleoparticles (mRNPs) in yeast. However, the molecular mechanisms of this nuclear pore-dependent mRNA quality control, including the sumoylated targets of Ulp1, have remained unidentified. Here, we demonstrate that the apparent 'pre-mRNA leakage' phenotype arising upon ULP1 inactivation is shared by sumoylation mutants of the THO complex, an early mRNP biogenesis factor. Importantly, we establish that alteration of THO complex activity differentially impairs the expression of intronless and intron-containing reporter genes, rather than triggering bona fide 'pre-mRNA leakage'. Indeed, we show that the presence of introns within THO target genes attenuates the effect of THO inactivation on their transcription. Epistasis analyses further clarify that different nuclear pore components influence intron-containing gene expression at distinct stages. Ulp1, whose maintenance at nuclear pores depends on the Nup84 complex, impacts on THO-dependent gene expression, whereas the nuclear basket-associated Mlp1/Pml39 proteins prevent pre-mRNA export at a later stage, contributing to mRNA quality control. Our study thus highlights the multiplicity of mechanisms by which nuclear pores contribute to gene expression, and further provides the first evidence that intronic sequences can alleviate early mRNP biogenesis defects

Natural variation in a neural globin tunes oxygen sensing in wild Caenorhabditis elegans.

(Note:A. Persson and E Gross contributed equally to this study.)info:eu-repo/semantics/publishe

GH Receptor Plays a Major Role in Liver Regeneration through the Control of EGFR and ERK1/2 Activation.

International audienceGH is a pleiotropic hormone that plays a major role in proliferation, differentiation, and metabolism via its specific receptor. It has been previously suggested that GH signaling pathways are required for normal liver regeneration but the molecular mechanisms involved have yet to be determined. The aim of this study was to identify the mechanisms by which GH controls liver regeneration. We performed two thirds partial hepatectomies in GH receptor (GHR)-deficient mice and wild-type littermates and showed a blunted progression in the G(1)/S transition phase of the mutant hepatocytes. This impaired liver regeneration was not corrected by reestablishing IGF-1 expression. Although the initial response to partial hepatectomy at the priming phase appeared to be similar between mutant and wild-type mice, cell cycle progression was significantly blunted in mutant mice. The main defect in GHR-deficient mice was the deficiency of the epidermal growth factor receptor activation during the process of liver regeneration. Finally, among the pathways activated downstream of GHR during G(1) phase progression, namely Erk1/2, Akt, and signal transducer and activator of transcription 3, we only found a reduced Erk1/2 phosphorylation in mutant mice. In conclusion, our results demonstrate that GH signaling plays a major role in liver regeneration and strongly suggest that it acts through the activation of both epidermal growth factor receptor and Erk1/2 pathways

Natural variation in a neural globin tunes oxygen sensing in wild Caenorhabditis elegans

(Note:A. Persson and E Gross contributed equally to this study.)info:eu-repo/semantics/publishe