GC content shapes mRNA storage and decay in human cells.

mRNA translation and decay appear often intimately linked although the rules of this interplay are poorly understood. In this study, we combined our recent P-body transcriptome with transcriptomes obtained following silencing of broadly acting mRNA decay and repression factors, and with available CLIP and related data. This revealed the central role of GC content in mRNA fate, in terms of P-body localization, mRNA translation and mRNA stability: P-bodies contain mostly AU-rich mRNAs, which have a particular codon usage associated with a low protein yield; AU-rich and GC-rich transcripts tend to follow distinct decay pathways; and the targets of sequence-specific RBPs and miRNAs are also biased in terms of GC content. Altogether, these results suggest an integrated view of post-transcriptional control in human cells where most translation regulation is dedicated to inefficiently translated AU-rich mRNAs, whereas control at the level of 5' decay applies to optimally translated GC-rich mRNAs

Ribosome-associated vesicles: A dynamic subcompartment of the endoplasmic reticulum in secretory cells

The endoplasmic reticulum (ER) is a highly dynamic network of membranes. Here, we combine live-cell microscopy with in situ cryo–electron tomography to directly visualize ER dynamics in several secretory cell types including pancreatic β-cells and neurons under near-native conditions. Using these imaging approaches, we identify a novel, mobile form of ER, ribosome-associated vesicles (RAVs), found primarily in the cell periphery, which is conserved across different cell types and species. We show that RAVs exist as distinct, highly dynamic structures separate from the intact ER reticular architecture that interact with mitochondria via direct intermembrane contacts. These findings describe a new ER subcompartment within cells

Etude du rôle des activateurs de transcription paralogues Aft1p et Aft2p dans la régulation du métabolisme du fer chez la levure Saccharomyces cerevisiae

Iron is an essential nutrient, but its accumulation can be highly cytotoxic. Iron homeostasis must be tightly regulated in order to satisfy cell demand without damage. In the yeast Saccharomyces cerevisiae, this regulation is mediated by two paralogous transcription factors: Aft1p and Aft2p. Under iron starvation conditions, Aft1p activates the transcription of high affinity iron transport related genes by specific binding to the DNA consensus sequence 5'-TGCACCC-3' found in the promoters of these genes. The role of Aft2p remains poorly understood. Aft2p is capable of binding in vitro to the Aft1p DNA consensus sequence and it activates the transcription of some of the Aft1p target gene under specific conditions, such as overexpression, or expression of a gain-of-function allele. We aimed at further understanding the role of Aft2p in a physiological context, by identification of its target genes and characterisation of its mode of action. For this purpose, we performed global transcriptome analyses, Northern blot and chromatin immunoprecipitation experiments using wild type and Δaft1 and/or Δaft2 mutant strains grown under iron depleted conditions. We demonstrated that Aft2p directly activates the transcription of genes that are essential for the iron intracellular sub-compartimentalization and use, while Aft1p does not. We discovered that the consensus sequence in the promoter region of genes specifically activated by At2p was not the 5'-TGCACCC-3' sequence, but the shorter 5'-(G/A)CACCC- 3'. Aft2p plays also a direct role in the transcriptional regulation of some genes involved in zinc metabolism, and it could be involved in the regulation of ergosterol and fatty acid metabolism. We also showed that Aft1p and Aft2p proteins amount is iron-regulated. The modulation of Aft1p protein amount can be attributed to its own transcriptional regulation, while the modulation of Aft2p protein amount involves post-transcriptional regulation.Le fer est à la fois indispensable et toxique pour les cellules. Un contrôle strict de son métabolisme est nécessaire pour pourvoir aux besoins des cellules tout en évitant ses effets délétères. Chez la levure Saccharomyces cerevisiae, ce contrôle est assuré par les activateurs transcriptionnels paralogues Aft1p et Aft2p. En condition de carence en fer, Aft1p active la transcription des gènes du transport à haute affinité du fer extracellulaire en se fixant sur la séquence cis-régulatrice 5'-TGCACCC-3'. Le rôle d'Aft2p est moins bien caractérisé ; il reconnaît in vitro la même séquence consensus qu'Aft1p et active la transcription de plusieurs gènes régulés par Aft1p lorsqu'il est surexprimé ou sous une forme mutée constitutivement active. Nous avons cherché à mieux comprendre le rôle d'Aft2p en condition de carence en fer en identifiant ses gènes cibles et en caractérisant son mode d'action, par une combinaison d'expériences d'analyses globales de transcriptome, de Northern blot et d'immunoprécipitation de la chromatine réalisées avec les souches sauvage et délétées d'AFT1 et/ou d'AFT2. Nous avons montré qu'Aft2p, mais pas Aft1p, active directement la transcription des gènes de l'utilisation et du stockage du fer intracellulaire. Nous avons mis en évidence que la séquence cis-régulatrice des gènes régulés par Aft2p n'est pas 5'-TGCACCC-3', mais une séquence plus courte, 5'-(G/A)CACCC-3'. Aft2p joue également un rôle direct dans la régulation transcriptionnelle de plusieurs gènes du métabolisme du zinc, et il pourrait intervenir dans la régulation des gènes du métabolisme de l'ergostérol et des acides gras. Par ailleurs, nous avons montré l'existence d'une régulation par le fer de la quantité des protéines Aft1p et Aft2p. La variation de quantité d'Aft1p semble résulter de son autorégulation transcriptionnelle, alors que la variation de quantité d'Aft2p implique une régulation post-transcriptionnelle

Etude du rôle des activateurs de transcription paralogues Aft1p et Aft2p dans la régulation du métabolisme du fer chez la levure Saccharomyces cerevisiae

Le fer est à la fois indispensable et toxique pour les cellules. Un contrôle strict de son métabolisme est nécessaire pour pourvoir aux besoins des cellules tout en évitant ses effets délétères. Chez la levure Saccharomyces cerevisiae, ce contrôle est assuré par les activateurs transcriptionnels paralogues Aft1p et Aft2p. En condition de carence en fer, Aft1p active la transcription des gènes du transport à haute affinité du fer extracellulaire en se fixant sur la séquence cis-régulatrice 5 -TGCACCC-3 . Le rôle d Aft2p est moins bien caractérisé ; il reconnaît in vitro la même séquence consensus qu Aft1p et active la transcription de plusieurs gènes régulés par Aft1p lorsqu il est surexprimé ou sous une forme mutée constitutivement active. Nous avons cherché à mieux comprendre le rôle d Aft2p en condition de carence en fer en identifiant ses gènes cibles et en caractérisant son mode d action, par une combinaison d expériences d analyses globales de transcriptome, de Northern blot et d immunoprécipitation de la chromatine réalisées avec les souches sauvage et délétées d AFT1 et/ou d AFT2. Nous avons montré qu Aft2p, mais pas Aft1p, active directement la transcription des gènes de l utilisation et du stockage du fer intracellulaire. Nous avons mis en évidence que la séquence cis-régulatrice des gènes régulés par Aft2p n est pas 5 -TGCACCC-3 , mais une séquence plus courte, 5 -(G/A)CACCC-3 . Aft2p joue également un rôle direct dans la régulation transcriptionnelle de plusieurs gènes du métabolisme du zinc, et il pourrait intervenir dans la régulation des gènes du métabolisme de l ergostérol et des acides gras. Par ailleurs, nous avons montré l existence d une régulation par le fer de la quantité des protéines Aft1p et Aft2p. La variation de quantité d Aft1p semble résulter de son autorégulation transcriptionnelle, alors que la variation de quantité d Aft2p implique une régulation post-transcriptionnelleIron is an essential nutrient, but its accumulation can be highly cytotoxic. Iron homeostasis must be tightly regulated in order to satisfy cell demand without damage. In the yeast Saccharomyces cerevisiae, this regulation is mediated by two paralogous transcription factors: Aft1p and Aft2p. Under iron starvation conditions, Aft1p activates the transcription of high affinity iron transport related genes by specific binding to the DNA consensus sequence 5 -TGCACCC-3 found in the promoters of these genes. The role of Aft2p remains poorly understood. Aft2p is capable of binding in vitro to the Aft1p DNA consensus sequence and it activates the transcription of some of the Aft1p target gene under specific conditions, such as overexpression, or expression of a gain-of-function allele. We aimed at further understanding the role of Aft2p in a physiological context, by identification of its target genes and characterisation of its mode of action. For this purpose, we performed global transcriptome analyses, Northern blot and chromatin immunoprecipitation experiments using wild type and aft1 and/or aft2 mutant strains grown under iron depleted conditions. We demonstrated that Aft2p directly activates the transcription of genes that are essential for the iron intracellular sub-compartimentalization and use, while Aft1p does not. We discovered that the consensus sequence in the promoter region of genes specifically activated by At2p was not the 5 -TGCACCC-3 sequence, but the shorter 5 -(G/A)CACCC- 3 . Aft2p plays also a direct role in the transcriptional regulation of some genes involved in zinc metabolism, and it could be involved in the regulation of ergosterol and fatty acid metabolism. We also showed that Aft1p and Aft2p proteins amount is iron-regulated. The modulation of Aft1p protein amount can be attributed to its own transcriptional regulation, while the modulation of Aft2p protein amount involves post-transcriptional regulationPARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Direct Activation of Genes Involved in Intracellular Iron Use by the Yeast Iron-Responsive Transcription Factor Aft2 without Its Paralog Aft1

The yeast Saccharomyces cerevisiae contains a pair of paralogous iron-responsive transcription activators, Aft1 and Aft2. Aft1 activates the cell surface iron uptake systems in iron depletion, while the role of Aft2 remains poorly understood. This study compares the functions of Aft1 and Aft2 in regulating the transcription of genes involved in iron homeostasis, with reference to the presence/absence of the paralog. Cluster analysis of DNA microarray data identified the classes of genes regulated by Aft1 or Aft2, or both. Aft2 activates the transcription of genes involved in intracellular iron use in the absence of Aft1. Northern blot analyses, combined with chromatin immunoprecipitation experiments on selected genes from each class, demonstrated that Aft2 directly activates the genes SMF3 and MRS4 involved in mitochondrial and vacuolar iron homeostasis, while Aft1 does not. Computer analysis found different cis-regulatory elements for Aft1 and Aft2, and transcription analysis using variants of the FET3 promoter indicated that Aft1 is more specific for the canonical iron-responsive element TGCACCC than is Aft2. Finally, the absence of either Aft1 or Aft2 showed an iron-dependent increase in the amount of the remaining paralog. This may provide additional control of cellular iron homeostasis

Genome-Wide Screen for Genes With Effects on Distinct Iron Uptake Activities in Saccharomyces cerevisiae

We screened a collection of 4847 haploid knockout strains (EUROSCARF collection) of Saccharomyces cerevisiae for iron uptake from the siderophore ferrioxamine B (FOB). A large number of mutants showed altered uptake activities, and a few turned yellow when grown on agar plates with added FOB, indicating increased intracellular accumulation of undissociated siderophores. A subset consisting of 197 knockouts with altered uptake was examined further for regulated activities that mediate cellular uptake of iron from other siderophores or from iron salts. Hierarchical clustering analysis grouped the data according to iron sources and according to mutant categories. In the first analysis, siderophores grouped together with the exception of enterobactin, which grouped with iron salts, suggesting a reductive pathway of iron uptake for this siderophore. Mutant groupings included three categories: (i) high-FOB uptake, high reductase, low-ferrous transport; (ii) isolated high- or low-FOB transport; and (iii) induction of all activities. Mutants with statistically altered uptake activities included genes encoding proteins with predominant localization in the secretory pathway, nucleus, and mitochondria. Measurements of different iron-uptake activities in the yeast knockout collection make possible distinctions between genes with general effects on iron metabolism and those with pathway-specific effects