Ex vivo correction of selenoprotein N deficiency in rigid spine muscular dystrophy caused by a mutation in the selenocysteine codon

Premature termination of translation due to nonsense mutations is a frequent cause of inherited diseases. Therefore, many efforts were invested in the development of strategies or compounds to selectively suppress this default. Selenoproteins are interesting candidates considering the idiosyncrasy of the amino acid selenocysteine (Sec) insertion mechanism. Here, we focused our studies on SEPN1, a selenoprotein gene whose mutations entail genetic disorders resulting in different forms of muscular diseases. Selective correction of a nonsense mutation at the Sec codon (UGA to UAA) was undertaken with a corrector tRNASec that was engineered to harbor a compensatory mutation in the anticodon. We demonstrated that its expression restored synthesis of a full-length selenoprotein N both in HeLa cells and in skin fibroblasts from a patient carrying the mutated Sec codon. Readthrough of the UAA codon was effectively dependent on the Sec insertion machinery, therefore being highly selective for this gene and unlikely to generate off-target effects. In addition, we observed that expression of the corrector tRNASec stabilized the mutated SEPN1 transcript that was otherwise more subject to degradation. In conclusion, our data provide interesting evidence that premature termination of translation due to nonsense mutations is amenable to correction, in the context of the specialized selenoprotein synthesis mechanism

cDNA Library Generation for the Analysis of Small RNAs by High-Throughput Sequencing

The RNome of a cell is highly diverse and consists besides messenger RNAs (mRNAs), transfer RNAs (tRNAs), and ribosomal RNAs (rRNAs) also of other small and long transcript entities without apparent coding potential. This class of molecules, commonly referred to as non-protein-coding RNAs (ncRNAs), is involved in regulating numerous biological processes and thought to contribute to cellular complexity. Therefore, much effort is put into their identification and further functional characterization. Here we provide a cost-effective and reliable method for cDNA library construction of small RNAs in the size range of 20-500 residues. The effectiveness of the described method is demonstrated by the analysis of ribosome-associated small RNAs in the eukaryotic model organism Trypanosoma brucei

Increased Muscle Stress-Sensitivity Induced by Selenoprotein N Inactivation in Mouse: A Mammalian Model for SEPN1-Related Myopathy

Selenium is an essential trace element and selenoprotein N (SelN) was the first selenium-containing protein shown to be directly involved in human inherited diseases. Mutations in the SEPN1 gene, encoding SelN, cause a group of muscular disorders characterized by predominant affection of axial muscles. SelN has been shown to participate in calcium and redox homeostasis, but its pathophysiological role in skeletal muscle remains largely unknown. To address SelN function in vivo, we generated a Sepn1-null mouse model by gene targeting. The Sepn1−/− mice had normal growth and lifespan, and were macroscopically indistinguishable from wild-type littermates. Only minor defects were observed in muscle morphology and contractile properties in SelN-deficient mice in basal conditions. However, when subjected to challenging physical exercise and stress conditions (forced swimming test), Sepn1−/− mice developed an obvious phenotype, characterized by limited motility and body rigidity during the swimming session, as well as a progressive curvature of the spine and predominant alteration of paravertebral muscles. This induced phenotype recapitulates the distribution of muscle involvement in patients with SEPN1-Related Myopathy, hence positioning this new animal model as a valuable tool to dissect the role of SelN in muscle function and to characterize the pathophysiological process

Understanding the importance of selenium and selenoproteins in muscle function

Selenium is an essential trace element. In cattle, selenium deficiency causes dysfunction of various organs, including skeletal and cardiac muscles. In humans as well, lack of selenium is associated with many disorders, but despite accumulation of clinical reports, muscle diseases are not generally considered on the list. The goal of this review is to establish the connection between clinical observations and the most recent advances obtained in selenium biology. Recent results about a possible role of selenium-containing proteins in muscle formation and repair have been collected. Selenoprotein N is the first selenoprotein linked to genetic disorders consisting of different forms of congenital muscular dystrophies. Understanding the muscle disorders associated with selenium deficiency or selenoprotein N dysfunction is an essential step in defining the causes of the disease and obtaining a better comprehension of the mechanisms involved in muscle formation and maintenance

Uncovering the Importance of Selenium in Muscle Disease

A connection between selenium bioavailability and development of muscular disorders both in humans and livestock has been established for a long time. With the development of genomics, the function of several selenoproteins was shown to be involved in muscle activity, including SELENON, which was linked to an inherited form of myopathy. Development of animal models has helped to dissect the physiological dysfunction due to mutation in the SELENON gene; however the molecular activity remains elusive and only recent analysis using both in vivo and in vitro experiment provided hints toward its function in oxidative stress defence and calcium transport control. This review sets out to summarise most recent findings for the importance of selenium in muscle function and the contribution of this information to the design of strategies to cure the diseases

ÉTUDE DU RÔLE DU SÉLÉNIUM ET DE LA SÉLÉNOPROTÉINE N<br />DANS LES PATHOLOGIES MUSCULAIRES.

2006-09-15Selenium was considered originally as a poison. It is only in the mid-fifties that the physiological significance of this trace element was correctly evaluated; identification of pathologies related to selenium deficiencies demonstrated its essential nutrient function in livestock and later in humans.Biochemical studies identified selenocysteine as the major biological form of selenium in animals and bacteria. This particular amino acid is specifically incorporated into selenoproteins through a dedicated translation machinery.Most of the known selenoproteins have not been attributed a function yet. Among them is selenoprotein N (SePN), a novel selenium containing protein identified in our laboratory in 1999. In 2001, it was shown that several muscular disorders segregate with a locus containing SEPN1, the SePN encoding gene. These diseases are now collectively classified as SePN-related myopathies. At the beginning of my PhD studies, little was known about selenoprotein N. To identify its function, several approaches were developed concomitantly.First, I contributed to demonstrate that SePN is a 65kDa trans-membrane glycoprotein localized within the endoplasmic reticulum. Then, several successive approaches allowed characterization of SePN interacting partners in the membrane whose identification and further validation are underway.In a second step, we developed two animal models for SePN related myopathies. Owing to an antisense strategy, it was shown that inhibition of SePN expression during zebrafish embryogenesis led to severe muscular defects and abnormal development. In parallel, taking advantage of the Cre-Lox system, we generated either the complete knock-out of the gene in mice or the conditional targeted disruption of the SEPN1 gene in muscle only. Both models showed no apparent defects. Histological studies, however, showed that muscles featured dystrophic muscular fiber patterns. In addition, we showed that SEPN1 knockout mice displayed increased sensitivity to induced oxidative stress. The functional exploration of these models will be pursued in collaboration with several groups.Finally, another project that was tackled consisted in engineering a correction strategy based on the use of a modified tRNASec to force recognition of a mutated Sec codon in a RSMD1 patient with a homozygous point mutation. Successful experiments conducted in cultured cells opened the route toward a possible gene therapy for these patients.Altogether, the studies will help obtaining a better insight into SePN function, especially in muscle physiology, as well as to increase our knowledge regarding selenium role in human health. The final aim of these studies is to develop targeted diagnostic and therapeutic tools that will derive from our predictive models.Longtemps considéré comme un composé toxique, le sélénium est maintenantlargement reconnu comme oligo-élément essentiel. Des carences alimentaires ont étéassociées à de nombreuses pathologies.La sélénocystéine est la forme biologique principale du sélénium. Cet acide aminé particulierest spécifiquement incorporé dans les sélénoprotéines grâce à une machinerie traductionnelledédiée en réponse à un codon UGA, traditionnellement reconnu comme un codon stop.A ce jour, la fonction moléculaire de la plupart des sélénoprotéines demeure inconnue. Parmicelles-ci figure la sélénoprotéine N (SePN), une nouvelle protéine à sélénium identifiée en1999 dans notre laboratoire par une approche bioinformatique.En 2001, il a été démontré que des mutations dans le gène SEPN1 codant pour SePN étaientresponsables de différentes pathologies musculaires regroupées dorénavant sous le terme demyopathies apparentées à la sélénoprotéine N.Au début de ma thèse, peu de choses étaient connues sur la fonction de SePN. Pourcomprendre son rôle, nous avons entrepris son étude selon différentes approches.Dans un premier temps, j'ai contribué à montrer que SePN est une glycoprotéine de 65kDa,associée aux membranes du réticulum endoplasmique. Ensuite, des approches biochimiquessuccessives ont permis de mettre en évidence son interaction avec différentes protéines de lamembrane, et dont l'identification est en cours.Dans un deuxième temps, nous avons mis au point deux modèles animaux des pathologiesmusculaires associées à un dysfonctionnement de SePN. Par une approche antisens, il a étéobservé que l'inhibition de l'expression de SePN au cours du développement embryonnairechez le poisson zèbre entraînait une altération de l'organisation du tissu musculaire.Parallèlement, tirant avantage du système Cre-Lox, nous avons obtenu des souris invalidéespour SEPN1 dans tout l'organisme ou de façon tissu spécifique dans le muscle. De façonsurprenante, les animaux ainsi obtenus ne présentent pas de phénotype apparent, même si lesanalyses histologiques préliminaires permettent d'observer un profil dystrophique classiquedes fibres musculaires. En outre, les animaux semblent présenter une sensibilité accrue austress oxydatif induit. L'exploration fonctionnelle de ce modèle est poursuivie au laboratoireet fait l'objet de plusieurs collaborations.Enfin, une autre étude entreprise au cours de ma thèse concerne une mutation pathologique dugène SEPN1 conduisant à l'apparition de myopathies chez l'homme. Par une approcheoriginale déduite du mécanisme atypique de traduction des sélénoprotéines, une stratégie quipourrait aboutir à terme à une thérapie génique pour certains patients a été mise au point.L'ensemble de ces travaux va permettre d'augmenter nos connaissances sur le rôle dela sélénoprotéine N dans le muscle ainsi que sur la fonction biologique de l'oligo-élémentsélénium dans ce tissu. Le but ultime de l'ensemble de ces travaux est de développer desoutils de diagnostic ainsi que des approches thérapeutiques ciblées