Étude de protéines liant les G-quadruplexes situés en 5’UTR de gènes impliqués dans la maladie de Parkinson

Au sein du système nerveux, les mécanismes de régulation post-transcriptionnelle contribuent grandement aux différents phénotypes cellulaires. En effet, de faibles dérégulations peuvent mener à des maladies. Les G-quadruplexes (G4) d’ARN émergent comme étant de nouveaux acteurs dans la régulation post-transcriptionnelle. Les G4 sont des structures secondaires très stables retrouvées dans l’ADN et l’ARN. Ils sont formés par l’empilement d’un minimum de deux tétrades de guanines et stabilisés par des cations monovalents, habituellement le potassium. Au niveau de l’ARN, ils sont impliqués dans différents mécanismes comme l’épissage alternatif, la polyadénylation alternative, l’activation et l’inhibition de la traduction et la maturation des microARN. Selon plusieurs, les G4 sont considérés comme étant des évènements plutôt que des entités. Des éléments trans, soit des protéines, sont alors en mesure d’assurer leur repliement et dépliement. Une analyse bio-informatique a confirmé un enrichissement de G4 d’ARN dans les transcrits du système nerveux. Cette analyse a aussi permis d’identifier la maladie de Parkinson comme l’une des maladies ayant la plus grande proportion de potentiel G4 (pG4). En effet, sur 16 gènes reliés à la maladie, 15 d’entre eux contiennent au moins un pG4. Ensuite, différentes techniques biochimiques comme des essais de fluorescence, le dichroïsme circulaire et la cartographie in-line ont été utilisées pour confirmer la formation de certains G4 situés dans des régions 5’ non traduites (5’UTR). Ces techniques ont permis d’identifier 4 nouveaux G4 d’ARN n’ayant jamais été caractérisés auparavant. Par la suite, des essais luciférase ont conclus que deux de ces G4, chez les ARNm de VPS35 et PRKN, ont un effet répresseur sur la traduction. Les protéines pouvant lier ces deux G4 ont ensuite été analysées par spectrométrie de masse. La protéine Guanine Nucleotide-Binding Protein-Like 1 (GNL1) a alors été identifiée comme étant une protéine pouvant lier les deux G4. Finalement, l’interaction entre GNL1 et le G4 de VPS35 et PRKN a été confirmée par des essais de retardement sur gel. En bref, cette étude a permis d’identifier une nouvelle protéine, GNL1, pouvant lier deux nouveaux G-quadruplexes se situant dans le 5'UTR de gènes dérégulés dans la maladie de Parkinson. Une régulation de l’interaction entre GNL1 et les G4 pourrait être une option thérapeutique pour contrer les dérégulations présentes dans la maladie de Parkinson.Within the nervous system, the post-transcriptional regulatory mechanisms contribute to the different cellular phenotypes. Indeed, weak deregulations can lead to diseases. RNA G-quadruplexes (G4) are emerging as new players in post-transcriptional regulation. G4s are very stable secondary structures found in DNA and RNA. They are formed by a minimum of two tetrads of guanines and stabilized by monovalent cations usually potassium. At the RNA level, they are involved in different mechanisms, such as alternative polyadenylation, alternative splicing, activation and inhibition of translation, and maturation of microRNAs. According to many, G4s are seen as events rather than entities. Trans elements, like proteins, are able to ensure their folding and unfolding. A bioinformatic analysis confirmed that there is indeed an enrichment of RNA G4s in the transcripts of the nervous system. This analysis also identified Parkinson's disease as one of the diseases with the highest proportion of potential G4 (pG4). Out of 16 disease-related genes, 15 of them contain a least one pG4. Then, different biochemical techniques such as fluorescence assay, circular dichroism, and in-line probing were used to confirm the formation of G4s located in the different 5’ untranslated regions (5’UTR). These techniques identified 4 new RNA G4s that have never been characterized before. Subsequently, luciferase assays concluded that two of these G4s, in the mRNA of VPS35 and PRKN, have a repressive effect on translation. The proteins capable of binding these two G4s were then identified by mass spectrometry. The protein Guanine Nucleotide-Binding Protein-Like 1 (GNL1) was identified as being a protein capable of binding the two G4s. Finally, the interaction between GNL1 and the G4 from VPS35 and PRKN was confirmed by electrophoretic mobility shift assays. In brief, this study identified a new protein, GNL1, which can bind two new G-quadruplexes located in deregulated genes associated with Parkinson's disease. The regulation of the interaction between GNL1 and the two G4s could be a therapeutic option to counter the deregulations present in Parkinson's disease

Étude du périderme de tubercules de somaclones de pomme de terre plus résistants à la gale commune

La pomme de terre est le 4e légume le plus consommé au monde et se retrouve au premier rang au Canada. D’ailleurs, ce pays est le 12e plus gros producteur de pommes de terre mondialement. Certaines maladies s’attaquent à cette culture de grande importance, comme la gale commune de la pomme de terre. Les meilleures méthodes de contrôle de la maladie sont des méthodes culturales (rotation des cultures, contrôle du pH, etc.) et variétales (culture de variétés naturellement plus résistantes). La bactérie responsable de la maladie, Streptomyces scabiei, synthétise une toxine essentielle pour l’apparition des symptômes, la thaxtomine A. Dans des projets antérieurs du laboratoire de Nathalie Beaudoin, des cals de pommes de terre de la variété Yukon Gold ont été habitués à des concentrations croissantes de thaxtomine A. Certains des somaclones régénérés à partir de ces cals sont plus résistants à la gale commune que la variété parentale. Le périderme de ces derniers a été caractérisé de plusieurs façons. Il a été possible de remarquer que celui-ci contient plus de couches de cellules subérisées que la variété parentale. La subérine induit la production de thaxtomine A par S. scabiei. La subérine contenue dans le périderme des somaclones habitués à la thaxomine A a été extraite et mise en culture avec S. scabiei. La production de thaxtomine A a ensuite été quantifée. Une baisse de production de TA lorsque S. scabiei est en contact avec la subérine extraite des somaclones habitués en comparant avec la subérine extraite de la variété parentale a été mesuré. De plus, il a également été possible de remarquer lors d’analyses par chromatographie en phase gazeuse-spectrométrie de masse (GC-MS) des différences dans la composition de la subérine insoluble des somaclones habitués comparée à la subérine de la variété parentale. Finalement, l’expression de plusieurs gènes reliés à la synthèse de la subérine et à la défense a été testé dans le périderme de mini-tubercules âgés de une à trois semaines. Tous les gènes étudiés ont montré une expression plus grande chez les somaclones habitués comparé à la variété parentale pour au moins une des trois semaines du test. Ces résultats ont pu être corrélés à la plus grande résistance des somaclones habitués à la gale commune. Les résultats de ce projet apportent une meilleure connaissance de la résistance à la gale commune, mais également des pistes pour la recherche de variétés plus résistantes qui pourraient être cultivées à grande échelle

Development of a highly optimized procedure for the discovery of RNA G-quadruplexes by combining several strategies

Abstract : RNA G-quadruplexes (rG4s) are non-canonical secondary structures that are formed by the selfassociation of guanine quartets and that are stabilized by monovalent cations (e.g. potassium). rG4s are key elements in several post-transcriptional regulation mechanisms, including both messenger RNA (mRNA) and microRNA processing, mRNA transport and translation, to name but a few examples. Over the past few years, multiple high-throughput approaches have been developed in order to identify rG4s, including bioinformatic prediction, in vitro assays and af nity capture experiments coupled to RNA sequencing. Each individual approach had its limits, and thus yielded only a fraction of the potential rG4 that are further con rmed (i.e., there is a signi cant level of false positive). This report aims to bene t from the strengths of several existing approaches to identify rG4s with a high potential of being folded in cells. Brie y, rG4s were pulled-down from cell lysates using the biotinylated biomimetic G4 ligand BioTASQ and the sequences thus isolated were then identi ed by RNA sequencing. Then, a novel bioinformatic pipeline that included DESeq2 to identify rG4 enriched transcripts, MACS2 to identify rG4 peaks, rG4-seq to increase rG4 formation probability and G4RNA Screener to detect putative rG4s was performed. This work ow uncovers new rG4 candidates whose rG4-folding was then con rmed in vitro using an array of established biophysical methods. Clearly, this work ow led to the identi cation of novel rG4s in a highly specic and reliable manner

Autonomous quantum error correction of Gottesman-Kitaev-Preskill states

The Gottesman-Kitaev-Preskill (GKP) code encodes a logical qubit into a bosonic system with resilience against single-photon loss, the predominant error in most bosonic systems. Here we present experimental results demonstrating quantum error correction of GKP states based on reservoir engineering of a superconducting device. Error correction is made autonomous through an unconditional reset of an auxiliary transmon qubit. The lifetime of the logical qubit is shown to be increased from quantum error correction, therefore reaching the point at which more errors are corrected than generated.Comment: 6 pages, 3 figures + 26 pages, 12 figure

Passive Mineral Carbonation of Mg-rich Mine Wastes by Atmospheric CO2

Mg-rich process tailings and waste rocks from mining operations can react spontaneously with atmospheric CO2 to form stable carbonate minerals by exothermic reactions. Over the last decade, we have conducted a number of laboratory and field experiments and surveys on both mine waste rocks and different types of mine tailings from Ni-Cu, chrysotile, and diamond mines. The experiments and surveys cover a wide range of time (103 to 108 s) and mass (1-108 g) scales. Mine waste rich in brucite or chrysotile enhances the mineral carbonation reactions. Water saturation, but more importantly, watering frequency, are highly important to optimize carbonation. Adjusting the chemical composition of the interstitial water to favour Mg dissolution and to prevent passivation of the reaction surfaces is crucial to ensure the progress of the carbonation reactions. Preservation of the permeability structure is also critical to facilitate water and CO2 migration in the rock wastes and tailings. In field experiments, CO2 supply controled by diffusion in the mining waste is slower than the reaction rate which limits the capture of atmospheric CO2. Industrial implementation of passive mineral carbonation of mine waste by atmospheric CO2 can be optimized using the above parameters

Guanine nucleotide-binding protein-like 1 (GNL1) binds RNA G-quadruplex structures in genes associated with Parkinson’s disease

Abstract : RNAs are highly regulated at the post-transcriptional level in neurodegenerative diseases and just a few mutations can significantly affect the fate of neuronal cells. To date, the impact of G-quadruplex (G4) regulation in neurodegenerative diseases like Parkinson’s disease (PD) has not been analysed. In this study, in silico potential G4s located in deregulated genes related to the nervous system were initially identified and were found to be significantly enriched. Several G4 sequences found in the 5ʹ untranslated regions (5ʹUTR) of mRNAs associated with Parkinson’s disease were demonstrated to in fact fold in vitro by biochemical assays. Subcloning of the full-length 5ʹUTRs of these candidates upstream of a luciferase reporter system led to the demonstration that the G4s of both Parkin RBR E3 Ubiquitin Protein Ligase (PRKN) and Vacuolar Protein Sorting-Associated Protein 35 (VPS35) significantly repressed the translation of both genes in SH-SY5Y cells. Subsequently, a strategy of using label-free RNA affinity purification assays with either of these two G4 sequences as bait isolated the Guanine Nucleotide- Binding Protein-Like 1 (GNL1). The latter was shown to have a higher affinity for the G4 sequences than for their mutated version. This study sheds light on new RNA G-quadruplexes located in genes dysregulated in Parkinson disease and a new G4-binding protein, GNL1

Development of a highly optimized procedure for the discovery of RNA G-quadruplexes by combining several strategies

International audienceRNA G-quadruplexes (rG4s) are non-canonical secondary structures that are formed by the selfassociation of guanine quartets and that are stabilized by monovalent cations (e.g. potassium). rG4s are key elements in several post-transcriptional regulation mechanisms, including both messenger RNA (mRNA) and microRNA processing, mRNA transport and translation, to name but a few examples. Over the past few years, multiple high-throughput approaches have been developed in order to identify rG4s, including bioinformatic prediction, in vitro assays and affinity capture experiments coupled to RNA sequencing. Each individual approach had its limits, and thus yielded only a fraction of the potential rG4 that are further confirmed (i.e., there is a significant level of false positive). This report aims to benefit from the strengths of several existing approaches to identify rG4s with a high potential of being folded in cells. Briefly, rG4s were pulled-down from cell lysates using the biotinylated biomimetic G4 ligand BioTASQ and the sequences thus isolated were then identified by RNA sequencing. Then, a novel bioinformatic pipeline that included DESeq2 to identify rG4 enriched transcripts, MACS2 to identify rG4 peaks, rG4-seq to increase rG4 formation probability and G4RNA Screener to detect putative rG4s was performed. This workflow uncovers new rG4 candidates whose rG4-folding was then confirmed in vitro using an array of established biophysical methods. Clearly, this workflow led to the identification of novel rG4s in a highly specific and reliable manner

CDK1-Mediated Phosphorylation of BAG3 Promotes Mitotic Cell Shape Remodeling and the Molecular Assembly of Mitotic p62 Bodies

The cochaperone BCL2-associated athanogene 3 (BAG3), in complex with the heat shock protein HSPB8, facilitates mitotic rounding, spindle orientation, and proper abscission of daughter cells. BAG3 and HSPB8 mitotic functions implicate the sequestosome p62/SQSTM1, suggesting a role for protein quality control. However, the interplay between this chaperone-assisted pathway and the mitotic machinery is not known. Here, we show that BAG3 phosphorylation at the conserved T285 is regulated by CDK1 and activates its function in mitotic cell shape remodeling. BAG3 phosphorylation exhibited a high dynamic at mitotic entry and both a non-phosphorylatable BAG3T285A and a phosphomimetic BAG3T285D protein were unable to correct the mitotic defects in BAG3-depleted HeLa cells. We also demonstrate that BAG3 phosphorylation, HSPB8, and CDK1 activity modulate the molecular assembly of p62/SQSTM1 into mitotic bodies containing K63 polyubiquitinated chains. These findings suggest the existence of a mitotically regulated spatial quality control mechanism for the fidelity of cell shape remodeling in highly dividing cells