Endoribonuclease L (RNase L) Regulates the Myogenic and Adipogenic Potential of Myogenic Cells

Skeletal muscle maintenance and repair involve several finely coordinated steps in which pluripotent stem cells are activated, proliferate, exit the cell cycle and differentiate. This process is accompanied by activation of hundreds of muscle-specific genes and repression of genes associated with cell proliferation or pluripotency. Mechanisms controlling myogenesis are precisely coordinated and regulated in time to allow the sequence of activation/inactivation of genes expression. Muscular differentiation is the result of the interplay between several processes such as transcriptional induction, transcriptional repression and mRNA stability. mRNA stability is now recognized as an essential mechanism of control of gene expression. For instance, we previously showed that the endoribonuclease L (RNase L) and its inhibitor (RLI) regulates MyoD mRNA stability and consequently muscle differentiation

M19 Modulates Skeletal Muscle Differentiation and Insulin Secretion in Pancreatic β-Cells through Modulation of Respiratory Chain Activity

Mitochondrial dysfunction due to nuclear or mitochondrial DNA alterations contributes to multiple diseases such as metabolic myopathies, neurodegenerative disorders, diabetes and cancer. Nevertheless, to date, only half of the estimated 1,500 mitochondrial proteins has been identified, and the function of most of these proteins remains to be determined. Here, we characterize the function of M19, a novel mitochondrial nucleoid protein, in muscle and pancreatic β-cells. We have identified a 13-long amino acid sequence located at the N-terminus of M19 that targets the protein to mitochondria. Furthermore, using RNA interference and over-expression strategies, we demonstrate that M19 modulates mitochondrial oxygen consumption and ATP production, and could therefore regulate the respiratory chain activity. In an effort to determine whether M19 could play a role in the regulation of various cell activities, we show that this nucleoid protein, probably through its modulation of mitochondrial ATP production, acts on late muscle differentiation in myogenic C2C12 cells, and plays a permissive role on insulin secretion under basal glucose conditions in INS-1 pancreatic β-cells. Our results are therefore establishing a functional link between a mitochondrial nucleoid protein and the modulation of respiratory chain activities leading to the regulation of major cellular processes such as myogenesis and insulin secretion

Caractérisation fonctionnelle de deux nouvelles protéines mitochondriales

Des approches bioinformatiques et d'hybridation d'ADNc à haute densité sur des gènes humains ont permis d'identifier des gènes potentiels préférentiellement exprimés dans le muscle strié humain et de fonction inconnue. Ce travail de thèse consiste en la caractérisation fonctionnelle de deux de ces gènes, appelés GENX-3587 et GENX-70612. Le GENX-3587 correspond au gène humain FEM1A, qui est un gène candidat pour le syndrome polykystique ovarien (PCOS), tandis que la protéine humaine FEM1A a été décrite comme participant à une voie de signalisation anti-inflammatoire médiée par la prostaglandine E2. Par l'élaboration d'un anticorps spécifique de FEM1A, nous avons montré pour la première fois que la protéine murine Fem1a est une protéine mitochondriale, exprimée préférentiellement dans des organes enrichis en mitochondries tels que le cerveau, le cœur et le foie. De manière intéressante, nous avons également observé que Fem1a est sur-exprimée dans les coeurs de souris ayant subi une ischémie, suggérant que Fem1a pourrait faire partie de la voie de signalisation cardioprotective de la prostaglandine E2 survenant après un infarctus du myocarde. Le GENX-70612 correspond au gène dénommé C6orf125 qui code pour une protéine de fonction inconnue, que nous avons dénommé mitochondrine. Nous avons démontré que la mitochondrine est une protéine mitochondriale exprimée préférentiellement au niveau du cerveau et du cœur de souris. De plus, nous avons montré que la mitochondrine participe au métabolisme énergétique de la mitochondrie en jouant un rôle positif sur la production d'ATP mitochondrial. Nous avons également montré que la mitochondrine est un régulateur positif essentiel de la différenciation musculaire tardive. Finalement, des expériences préliminaires semblent indiquer que la mitochondrine joue un rôle dans la sécrétion d'insuline par les cellules b-pancréatiques.Bioinformatical and high density cDNA hybridization studies on human genes have allowed to identify potential genes preferentially expressed in human striated muscle and of unknown function. This thesis work consists in the functional characterization of two of these genes : GENX-3587 and GENX-70612. GENX-3587 corresponds to the human FEM1A gene, which is a candidate gene for the polycystic ovary syndrome (PCOS), while the human protein FEM1A is involved in an anti-inflammatory signaling pathway mediated by prostaglandin E2. Generation of a FEM1A specific antibody allowed us to show for the first time that mouse Fem1a is a mitochondrial protein, which is preferentially expressed in organs enriched in mitochondria such as brain, heart and liver. Interestingly, we have also observed that Fem1a expression is up-regulated in hearts from mice subjected to ischemia injury, suggesting that Fem1a may be part of the prostaglandin E2 cardioprotective pathway occurring after myocardial infarction. GENX-70612 corresponds to a gene called C6orf125 encoding a protein of unknown function, we have called mitochondrin. We have demonstrated that mitochondrin is a mitochondrial protein preferentially expressed in the brain and in the heart of mice. Furthermore, we have shown that mitochondrin is involved in the energetic metabolism of mitochondria by playing a positive role on mitochondrial ATP production. We have also shown that mitochondrin is an essential positive regulator of late muscle differentiation. Finally, preliminary experiments seem to indicate that mitochondrin plays a role on insulin secretion from b-pancreatic cellsMONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Nuclear translocation of the cytoskeleton-associated protein, smALP, upon induction of skeletal muscle differentiation.

International audienceThe skALP isoform has been shown to play a critical role in actin organization and anchorage within the Z-discs of skeletal muscles, but no data is available on the function of the smALP isoform in skeletal muscle cells. Here, we show that upon induction of differentiation a nuclear translocation of smALP from the cytoplasm to the nucleus of C2C12 myoblasts, concomitant to an up-regulation of the protein expression, occurs in parallel with the nuclear accumulation of myogenin. Moreover, we demonstrate that the LIM domain of smALP is essential for the nuclear translocation of the protein

Fem1a is a mitochondrial protein up-regulated upon ischemia-reperfusion injury.

International audienceVarious expression studies have shown a preferential muscle expression of the mouse Fem1a gene, but no data is available on the subcellular localization of the corresponding protein. Here, using a specific antibody, we show that Fem1a is expressed preferentially in cardiac muscle, brain and liver. Moreover, using immunofluorescence and electron microscopy, as well as biochemical assays, we demonstrate that Fem1a is localized within mitochondria of C2C12 myoblasts and cardiac muscle cells. Finally, we show that the expression of Fem1a, which is a cellular partner of the EP4 receptor for prostaglandin E(2), is increased in mouse hearts after myocardial infarction

Y RNA fragment in extracellular vesicles confers cardioprotection via modulation of IL‐10 expression and secretion

Abstract Cardiosphere‐derived cells (CDCs) reduce myocardial infarct size via secreted extracellular vesicles (CDC‐EVs), including exosomes, which alter macrophage polarization. We questioned whether short non‐coding RNA species of unknown function within CDC‐EVs contribute to cardioprotection. The most abundant RNA species in CDC‐EVs is a Y RNA fragment (EV‐YF1); its relative abundance in CDC‐EVs correlates with CDC potency in vivo. Fluorescently labeled EV‐YF1 is actively transferred from CDCs to target macrophages via CDC‐EVs. Direct transfection of macrophages with EV‐YF1 induced transcription and secretion of IL‐10. When cocultured with rat cardiomyocytes, EV‐YF1‐primed macrophages were potently cytoprotective toward oxidatively stressed cardiomyocytes through induction of IL‐10. In vivo, intracoronary injection of EV‐YF1 following ischemia/reperfusion reduced infarct size. A fragment of Y RNA, highly enriched in CDC‐EVs, alters Il10 gene expression and enhances IL‐10 protein secretion. The demonstration that EV‐YF1 confers cardioprotection highlights the potential importance of diverse exosomal contents of unknown function, above and beyond the usual suspects (e.g., microRNAs and proteins)

Reduced insulin secretion in M19-deficient INS-1 cells.

<p>(<b>A</b>) Northern blot analysis of the <i>M19</i> gene in human tissues. Pa: pancreas; Ki: kidney; Sk: skeletal muscle; Li: liver; Lu: lung; Pl: placenta; Br: brain; He: heart. Molecular markers are shown on the left. (<b>B</b>) Fluorescence microscopy of INS-1 cells double-labeled with the M19-specific P70612 antibody (M19, merge; green) and the MitoTracker dye (MitoTracker, merge; red). (<b>C</b>) Cell fractionation of INS-1 cells. Proteins of the total cell lysate (Lys), the cytosolic (Cyt) and the mitochondria (Mi) fractions were subjected to Western immunobloting. The cytosolic protein tubulin, the mitochondrial protein VDAC and M19 are detected. (<b>D</b>) INS-1 cells were transfected with a control pHYPER vector (sh control) or with the pHYPER vector encoding a M19-specific shRNA (sh M19). Western immunoblot analysis of the cell extracts shows expression levels of M19 and the control protein, tubulin. ATP production was determined in these cells (<b>E</b>), and insulin secretion was measured under basal glucose conditions (<b>F</b>). Results are the mean ± SEM of five (<b>E</b>), or four (<b>F</b>) independent experiments. (*) indicates statistical significance at p<0.05.</p