Hakai is required for stabilization of core components of the m6A mRNA methylation machinery

N6-methyladenosine (m6A) is the most abundant internal modification on mRNA which influences most steps of mRNA metabolism and is involved in several biological functions. The E3 ubiquitin ligase Hakai was previously found in complex with components of the m6A methylation machinery in plants and mammalian cells but its precise function remained to be investigated. Here we show that Hakai is a conserved component of the methyltransferase complex in Drosophila and human cells. In Drosophila, its depletion results in reduced m6A levels and altered m6A-dependent functions including sex determination. We show that its ubiquitination domain is required for dimerization and interaction with other members of the m6A machinery, while its catalytic activity is dispensable. Finally, we demonstrate that the loss of Hakai destabilizes several subunits of the methyltransferase complex, resulting in impaired m6A deposition. Our work adds functional and molecular insights into the mechanism of the m6A mRNA writer complex

Functions and mechanisms of m6A mRNA modification in Drosophila melanogaster

In all kingdoms of life, cellular RNAs are naturally decorated with a great number of different chemical modifications that constitute the so-called epitranscriptome. Like epigenetic DNA and histone modifications, RNA modifications represent one of the important mechanisms of precise gene expression regulation in living organisms. The majority of known RNA modifications are found on abundant non- coding RNAs, such as ribosomal RNA and transfer RNA, nonetheless messenger RNA (mRNA) is also modified by a dozen different chemical marks. m6A (N6-methyladenosine) is the most abundant internal modifications on eukaryotic mRNA and is regulated by three classes of proteins: methyltransferases or “writers”, demethylases or “erasers” and m6A-binding proteins or “readers”. Through its effects on mRNA structure, localization, splicing, stability, and translation, m6A influences many biological processes, including stem cell renewal and differentiation, brain function, immunity, and cancer progression. Over the last decade, technical progress in biochemical detection methods together with countless studies in different model organisms made m6A the best-characterized epitranscriptomic mark so far. Nevertheless, our understanding of m6A biogenesis, molecular functions as well as biological relevance is far from being complete. The main aim of this PhD thesis was to expand our knowledge of this new layer of gene expression regulation with a focus on specific aspects of the m6A pathway that are still poorly investigated in Drosophila melanogaster. Molecular biology techniques along with transcriptomic and proteomic analysis were used to characterize components of the m6A writer complex and to explore the function of well-known as well as potential new m6A readers. Gene-specific knockout and gain-of-function transgenic fly lines were generated using the CRISPR-Cas9 system to study m6A functions in vivo. Our results showed that Hakai is a conserved core component of the m6A methyltransferase complex and plays a key role in the stabilization of other subunits (Vir, Fl(2)d and Flacc) of the m6A writing machinery independently of its enzymatic activity. Characterization of the m6A-binding proteins revealed that Ythdf is the main m6A reader in the nervous system of flies and directly interacts with Fmr1 to guide its mRNA target selection. Mechanistically, we showed that the m6A pathway controls larval NMJ morphology primarily via the Ythdf/Fmr1-mediated translational repression of the synaptic microtubule regulator futsch. In addition, we found that the uncharacterized RNA helicase Dhx57 preferentially binds to m6A- modified RNA in the GGACU sequence context independently from Ythdf and that Dhx57 mutants mimic the NMJ overgrowth phenotype of most m6A mutants. Although the m6A erasers FTO and ALKBH5 are found only in vertebrate species, other members of the same family of demethylases are conserved in flies. By exploring functions and targets of the least characterized member of the ALKBH family, we showed that Alkbh6 expression increases in response to different types of abiotic stress and its loss makes flies hypersensitive to oxidative stress and starvation. Novel findings presented in this study substantially improved our understanding of the composition of the m6A writer machinery as well as highlighted the importance of this modification for proper neuronal development of flies. In addition, we created an important resource of data and tools for further characterization of Dhx57 as potential new m6A reader and Alkbh6 as important player in the cellular response to stress. -- Dans tous les règnes du vivant, les ARN sont naturellement décorés d'un grand nombre de modifications chimiques qui constituent ce que l'on appelle l'épitranscriptome. Comme les modifications épigénétiques de l'ADN et des histones, les modifications de l'ARN représentent l'un des nombreux mécanismes de régulation de l'expression génétique dont dépendent les organismes vivants. La majorité des modifications connues de l'ARN se trouvent sur des ARN non codant abondants, tels que les ARN ribosomaux et les ARN de transfert, mais les ARN messagers (ARNm) sont également modifiés par une douzaine de marques chimiques différentes. m6A (N6-méthyladénosine) est la modification interne la plus abondante sur l'ARNm eucaryote et elle est régulée par trois classes de protéines: les méthyltransférases ou "writers", les déméthylases ou "erasers" et les protéines de liaison à la m6A ou "readers". Par ses effets sur la structure, la localisation, l'épissage, la stabilité et la traduction de l'ARNm, m6A influence de nombreux processus biologiques, notamment le renouvellement et la différenciation des cellules souches, les fonctions cérébrales, l'immunité et la progression du cancer. Au cours de la dernière décennie, les progrès techniques réalisés dans les méthodes de détection biochimique ainsi que les innombrables études dans différents organismes modèles ont fait de m6A la marque épitranscriptomique la mieux caractérisée à ce jour. Néanmoins, notre compréhension de la biogenèse de m6A, de ses fonctions moléculaires et de sa pertinence biologique est loin d'être complète. L'objectif principal de cette thèse de doctorat était d'élargir nos connaissances sur cette nouvelle dimension de régulation de l'expression génétique en se concentrant sur des aspects spécifiques de la voie m6A qui sont encore peu étudiés dans l'organisme modèle Drosophila melanogaster. Des techniques de biologie moléculaire ainsi que des analyses transcriptomiques et protéomiques ont été utilisées pour caractériser les composants du complexe de déposition de m6A et pour explorer la fonction de readers connus de m6A ainsi que de potentiels nouveaux readers de cette modification. Des lignées de mouches transgéniques de knock-out et de surexpression spécifiques de gènes ont été générées à l'aide du système CRISPR-Cas9 afin d'étudier les fonctions de m6A in vivo. Nos résultats ont montré que Hakai est un composant central conservé du complexe méthyltransférase m6A et qu'il joue un rôle clé dans la stabilisation d'autres sous-unités de la machinerie d'écriture m6A (Vir, Fl(2)d et Flacc), indépendamment de son activité enzymatique. La caractérisation des protéines liant m6A a révélé que Ythdf est le principal reader de m6A dans le système nerveux des mouches et qu'il interagit directement avec Fmr1 pour guider la sélection de l'ARNm cible. D'un point de vue mécanistique, nous avons montré que la voie m6A contrôle la morphologie de la jonction neuromusculaire (NMJ) larvaire principalement via la répression traductionnelle du régulateur des microtubules synaptiques futsch médiée par Ythdf/Fmr1. En outre, nous avons découvert que l'hélicase ARN non caractérisée Dhx57 se lie préférentiellement à l'ARN modifié par m6A dans le contexte de séquence GGACU indépendamment de Ythdf et que les mutants Dhx57 présentent un phénotype de surcroissance de la NMJ similaire à celui observé dans la plupart des mutants m6A. Bien que les erasers de m6A FTO et ALKBH5 ne soient présents que chez les vertébrés, d'autres membres de la même famille de déméthylases sont conservés chez les mouches. En explorant les fonctions et les cibles du membre le moins caractérisé de la famille ALKBH, nous avons montré que l'expression d'Alkbh6 augmente en réponse à différents types de stress abiotiques et que sa perte rend les mouches hypersensibles au stress oxydatif et jeûne. Les nouveaux résultats présentés dans cette étude ont considérablement amélioré notre compréhension de la composition de la machinerie d'écriture de la m6A et ont mis en évidence l'importance de cette modification pour le bon développement neuronal des mouches. En outre, nous avons créé une ressource importante de données et d'outils pour une caractérisation plus poussée de Dhx57 en tant que nouveau reader potentiel de m6A et d'Alkbh6 en tant qu'acteur important de la réponse cellulaire au stress

Arc 3′ UTR Splicing Leads to Dual and Antagonistic Effects in Fine-Tuning Arc Expression Upon BDNF Signaling

Activity-regulated cytoskeletal associated protein (Arc) is an immediate-early gene critically involved in synaptic plasticity and memory consolidation. Arc mRNA is rapidly induced by synaptic activation and a portion is locally translated in dendrites where it modulates synaptic strength. Being an activity-dependent effector of homeostatic balance, regulation of Arc is uniquely tuned to result in short-lived bursts of expression. Cis-Acting elements that control its transitory expression post-transcriptionally reside primarily in Arc mRNA 3′ UTR. These include two conserved introns which distinctively modulate Arc mRNA stability by targeting it for destruction via the nonsense mediated decay pathway. Here, we further investigated how splicing of the Arc mRNA 3′ UTR region contributes to modulate Arc expression in cultured neurons. Unexpectedly, upon induction with brain derived neurotrophic factor, translational efficiency of a luciferase reporter construct harboring Arc 3′ UTR is significantly upregulated and this effect is dependent on splicing of Arc introns. We find that, eIF2α dephosphorylation, mTOR, ERK, PKC, and PKA activity are key to this process. Additionally, CREB-dependent transcription is required to couple Arc 3′ UTR-splicing to its translational upregulation, suggesting the involvement of de novo transcribed trans-acting factors. Overall, splicing of Arc 3′ UTR exerts a dual and unique effect in fine-tuning Arc expression upon synaptic signaling: while inducing mRNA decay to limit the time window of Arc expression, it also elicits translation of the decaying mRNA. This antagonistic effect likely contributes to the achievement of a confined yet efficient burst of Arc protein expression, facilitating its role as an effector of synapse-specific plasticity

Free volume and dynamics in a lipid bilayer

The lateral diffusion of lipids and of small molecules inside a membrane is strictly related to the arrangement of acyl chains and to their mobility. In this study, we use FTIR and time resolved 2D-IR spectroscopic techniques to characterize the structure and dynamics of the hydrophobic region of palmitoyl-oleylphosphatidylcholine/cholesterol vesicles dispersed in water/dimethylsulfoxide solutions. By means of a non-polar probe, hexacarbonyl tungsten, we monitor the distribution of free volumes inside the bilayer and the conformational dynamics of hydrophobic tails in relation to the different compositions of the membrane or the different compositions of the solvent. Despite the important structural changes induced by the presence of DMSO in the solvating medium, the picosecond dynamics of the membrane is preserved under the different conditions

Arc 3ʹ UTR splicing leads to dual and antagonistic effects in fine-tuning arc expression upon BDNF signaling

Activity-regulated cytoskeletal associated protein (Arc) is an immediate-early gene critically involved in synaptic plasticity and memory consolidation. Arc mRNA is rapidly induced by synaptic activation and a portion is locally translated in dendrites where it modulates synaptic strength. Being an activity-dependent effector of homeostatic balance, regulation of Arc is uniquely tuned to result in short-lived bursts of expression. Cis-Acting elements that control its transitory expression post-transcriptionally reside primarily in Arc mRNA 3′ UTR. These include two conserved introns which distinctively modulate Arc mRNA stability by targeting it for destruction via the nonsense mediated decay pathway. Here, we further investigated how splicing of the Arc mRNA 3′ UTR region contributes to modulate Arc expression in cultured neurons. Unexpectedly, upon induction with brain derived neurotrophic factor, translational efficiency of a luciferase reporter construct harboring Arc 3′ UTR is significantly upregulated and this effect is dependent on splicing of Arc introns. We find that, eIF2α dephosphorylation, mTOR, ERK, PKC, and PKA activity are key to this process. Additionally, CREB-dependent transcription is required to couple Arc 3′ UTR-splicing to its translational upregulation, suggesting the involvement of de novo transcribed trans-acting factors. Overall, splicing of Arc 3′ UTR exerts a dual and unique effect in fine-tuning Arc expression upon synaptic signaling: while inducing mRNA decay to limit the time window of Arc expression, it also elicits translation of the decaying mRNA. This antagonistic effect likely contributes to the achievement of a confined yet efficient burst of Arc protein expression, facilitating its role as an effector of synapse-specific plasticity

Lamotrigine rescues neuronal alterations and prevents seizure-induced memory decline in an Alzheimer's disease mouse model

Epilepsy is a comorbidity associated with Alzheimer's disease (AD), often starting many years earlier than memory decline. Investigating this association in the early pre-symptomatic stages of AD can unveil new mechanisms of the pathology as well as guide the use of antiepileptic drugs to prevent or delay hyperexcitability-related pathological effects of AD. We investigated the impact of repeated seizures on hippocampal memory and amyloid-β (Aβ) load in pre-symptomatic Tg2576 mice, a transgenic model of AD. Seizure induction caused memory deficits and an increase in oligomeric Aβ42 and fibrillary species selectively in pre-symptomatic transgenic mice, and not in their wildtype littermates. Electrophysiological patch-clamp recordings in ex vivo CA1 pyramidal neurons and immunoblots were carried out to investigate the neuronal alterations associated with the behavioral outcomes of Tg2576 mice. CA1 pyramidal neurons exhibited increased intrinsic excitability and lower hyperpolarization-activated Ih current. CA1 also displayed lower expression of the hyperpolarization-activated cyclic nucleotide-gated HCN1 subunit, a protein already identified as downregulated in the AD human proteome. The antiepileptic drug lamotrigine restored electrophysiological alterations and prevented both memory deficits and the increase in extracellular Aβ induced by seizures. Thus our study provides evidence of pre-symptomatic hippocampal neuronal alterations leading to hyperexcitability and associated with both higher susceptibility to seizures and to AD-specific seizure-induced memory impairment. Our findings also provide a basis for the use of the antiepileptic drug lamotrigine as a way to counteract acceleration of AD induced by seizures in the early phases of the pathology

Image_1_Arc 3′ UTR Splicing Leads to Dual and Antagonistic Effects in Fine-Tuning Arc Expression Upon BDNF Signaling.PDF

<p>Activity-regulated cytoskeletal associated protein (Arc) is an immediate-early gene critically involved in synaptic plasticity and memory consolidation. Arc mRNA is rapidly induced by synaptic activation and a portion is locally translated in dendrites where it modulates synaptic strength. Being an activity-dependent effector of homeostatic balance, regulation of Arc is uniquely tuned to result in short-lived bursts of expression. Cis-Acting elements that control its transitory expression post-transcriptionally reside primarily in Arc mRNA 3′ UTR. These include two conserved introns which distinctively modulate Arc mRNA stability by targeting it for destruction via the nonsense mediated decay pathway. Here, we further investigated how splicing of the Arc mRNA 3′ UTR region contributes to modulate Arc expression in cultured neurons. Unexpectedly, upon induction with brain derived neurotrophic factor, translational efficiency of a luciferase reporter construct harboring Arc 3′ UTR is significantly upregulated and this effect is dependent on splicing of Arc introns. We find that, eIF2α dephosphorylation, mTOR, ERK, PKC, and PKA activity are key to this process. Additionally, CREB-dependent transcription is required to couple Arc 3′ UTR-splicing to its translational upregulation, suggesting the involvement of de novo transcribed trans-acting factors. Overall, splicing of Arc 3′ UTR exerts a dual and unique effect in fine-tuning Arc expression upon synaptic signaling: while inducing mRNA decay to limit the time window of Arc expression, it also elicits translation of the decaying mRNA. This antagonistic effect likely contributes to the achievement of a confined yet efficient burst of Arc protein expression, facilitating its role as an effector of synapse-specific plasticity.</p

Complex Dynamical Aspects of Organic Electrolyte Solutions

The molecular dynamics of acetone alkali halide (LiBr and LiI) solutions at different compositions, from the pure solvent up to the solubility limit and at different temperatures, from 5.5 to 44.6 \ub0C, was studied using depolarized Rayleigh scattering (DRS) and low-frequency Raman spectroscopy, which probe dynamical fluctuations of the polarizability anisotropy of the system. With the increase of salt concentration an overall slowing down of the picosecond relaxation was observed because of the constraining imposed by the cation on the orientational mobility of the solvent. In particular, two distinct relaxation processes were detected: a slower process (tens of picosecond) due to acetone molecules within the Li+ solvation shell and a fast one (units of picosecond) due to bulk-like acetone. The retardation effect on the global solvent relaxation is larger in LiI then LiBr solutions owing to the larger degree of ion-ion association (ion pairs) in the latter. The modulation of cation-solvent (ion\u2013dipole) interactions induced by cation-anion (ion\u2013ion) ones affects the solvent mobility and explains, on a molecular basis, viscosity trends. Overall, a correlation between fast solvation dynamics and ion association propensity was found. Furthermore, analysis of the spectral susceptibility \uf063\u201d(\uf06e\u303) evidences that the enhancement of Li+\u2026O=C contacts, largely responsible for electrostriction phenomena in these ionic solutions, also modifies the ultrafast librational motion of acetone molecules localized in the ionic solvation shell