RESUMO
Cellular response to redox imbalance is crucial for organismal health. microRNAs are implicated in stress responses. ALG-1, the C. elegans ortholog of human AGO2, plays an essential role in microRNA processing and function. Here we investigated the mechanisms governing ALG-1 expression in C. elegans and the players controlling lifespan and stress resistance downstream of ALG-1. We show that upregulation of ALG-1 is a shared feature in conditions linked to increased longevity (e.g., germline-deficient glp-1 mutants). ALG-1 knockdown reduces lifespan and oxidative stress resistance, while overexpression enhances survival against pro-oxidant agents but not heat or reductive stress. R02D3.7 represses alg-1 expression, impacting oxidative stress resistance at least in part via ALG-1. microRNAs upregulated in glp-1 mutants (miR-87-3p, miR-230-3p, and miR-235-3p) can target genes in the protein disulfide isomerase pathway and protect against oxidative stress. This study unveils a tightly regulated network involving transcription factors and microRNAs which controls organisms' ability to withstand oxidative stress.
Assuntos
Proteínas de Caenorhabditis elegans , MicroRNAs , Animais , Humanos , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Estresse Oxidativo/genética , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Intertissue RNA transport recently emerged as a novel signaling mechanism. In mammals, mounting evidence suggests that small RNA transfer between cells is widespread and used in various physiological contexts. In the nematode C. elegans, a similar mechanism is conferred by the systemic RNAi pathway. Members of the Systemic RNA Interference Defective (SID) family act at different steps of cellular RNA uptake and export. The limiting step in systemic RNA interference (RNAi) is the import of extracellular RNAs via the conserved double-stranded (dsRNA)-gated dsRNA channel SID-1. To better understand the role of RNAs as intertissue signaling molecules, we modified the function of SID-1 in specific tissues of C. elegans. We observed that sid-1 loss-of-function mutants are as healthy as wild-type worms. Conversely, overexpression of sid-1 in C. elegans intestine, muscle, or neurons rendered worms short-lived. The effects of intestinal sid-1 overexpression were attenuated by silencing the components of systemic RNAi sid-1, sid-2 and sid-5, implicating systemic RNA signaling in the lifespan reduction. Accordingly, tissue-specific overexpression of sid-2 and sid-5 also reduced worm lifespan. Additionally, an RNAi screen for components of several non-coding RNA pathways revealed that silencing the miRNA biogenesis proteins PASH-1 and DCR-1 rendered the lifespan of worms with intestinal sid-1 overexpression similar to controls. Collectively, our data support the notion that systemic RNA signaling must be tightly regulated, and unbalancing that process provokes a reduction in lifespan. We termed this phenomenon Intercellular/Extracellular Systemic RNA imbalance (InExS).
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Interferência de RNA , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Longevidade/genética , RNA de Cadeia Dupla/metabolismo , Proteínas de Membrana/genética , Mamíferos/genéticaRESUMO
DICER is a key enzyme in microRNA (miRNA) biogenesis. Here we show that aerobic exercise training up-regulates DICER in adipose tissue of mice and humans. This can be mimicked by infusion of serum from exercised mice into sedentary mice and depends on AMPK-mediated signaling in both muscle and adipocytes. Adipocyte DICER is required for whole-body metabolic adaptations to aerobic exercise training, in part, by allowing controlled substrate utilization in adipose tissue, which, in turn, supports skeletal muscle function. Exercise training increases overall miRNA expression in adipose tissue, and up-regulation of miR-203-3p limits glycolysis in adipose under conditions of metabolic stress. We propose that exercise training-induced DICER-miR-203-3p up-regulation in adipocytes is a key adaptive response that coordinates signals from working muscle to promote whole-body metabolic adaptations.
Assuntos
Tecido Adiposo/metabolismo , RNA Helicases DEAD-box/metabolismo , Exercício Físico/fisiologia , Ribonuclease III/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Adaptação Fisiológica/fisiologia , Adipócitos/metabolismo , Animais , Células Cultivadas , RNA Helicases DEAD-box/deficiência , RNA Helicases DEAD-box/genética , Feminino , Glicólise , Humanos , Masculino , Camundongos , Camundongos Knockout , MicroRNAs/genética , MicroRNAs/metabolismo , Condicionamento Físico Animal , Ribonuclease III/deficiência , Ribonuclease III/genéticaRESUMO
In the presence of galactose, lithium ions activate the unfolded protein response (UPR) by inhibiting phosphoglucomutase activity and causing the accumulation of galactose-related metabolites, including galactose-1-phosphate. These metabolites also accumulate in humans who have the disease classic galactosemia. Here, we demonstrate that Saccharomyces cerevisiae yeast strains harboring a deletion of UBX4, a gene encoding a partner of Cdc48p in the endoplasmic reticulum-associated degradation (ERAD) pathway, exhibit delayed UPR activation after lithium and galactose exposure because the deletion decreases galactose-1-phosphate levels. The delay in UPR activation did not occur in yeast strains in which key ERAD or proteasomal pathway genes had been disrupted, indicating that the ubx4Δ phenotype is ERAD-independent. We also observed that the ubx4Δ strain displays decreased oxygen consumption. The inhibition of mitochondrial respiration was sufficient to diminish galactose-1-phosphate levels and, consequently, affects UPR activation. Finally, we show that the deletion of the AMP-activated protein kinase ortholog-encoding gene SNF1 can restore the oxygen consumption rate in ubx4Δ strain, thereby reestablishing galactose metabolism, UPR activation, and cellular adaption to lithium-galactose challenge. Our results indicate a role for Ubx4p in yeast mitochondrial function and highlight that mitochondrial and endoplasmic reticulum functions are intertwined through galactose metabolism. These findings also shed new light on the mechanisms of lithium action and on the pathophysiology of galactosemia.
Assuntos
Galactose/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lítio/farmacologia , Mitocôndrias/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Retículo Endoplasmático/metabolismo , Galactose/metabolismo , Galactosefosfatos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Consumo de Oxigênio , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Splicing de RNA , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
RNA interference (RNAi) is a valuable technique to determine gene function. In Caenorhabditis elegans, RNAi can be achieved by feeding worms bacteria carrying a plasmid expressing double-stranded RNA (dsRNA) targeting a gene of interest. The most commonly used plasmid vector for this purpose is L4440. However, it has been noticed that sequences within L4440 may elicit unspecific effects. Here, we provide a comprehensive characterization of these effects and their mechanisms and describe new unexpected phenotypes uncovered by the administration of unspecific exogenous dsRNA. An example involves dsRNA produced by the multiple cloning site (MCS) of L4440, which shares complementary sequences with some widely used reporter vectors and induces partial transgene silencing via the canonical and antiviral RNAi pathway. Going beyond transgene silencing, we found that the reduced embryonic viability of mir-35-41(gk262) mutants is partially reversed by exogenous dsRNA via a mechanism that involves canonical RNAi. These results indicate cross-regulation between different small RNA pathways in C. elegans to regulate embryonic viability. Recognition of the possible unspecific effects elicited by RNAi vectors is important for rigorous interpretation of results from RNAi-based experiments.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Interferência de RNA , RNA de Cadeia Dupla/genética , RNA Interferente Pequeno/genética , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Embrião não Mamífero , Escherichia coli/genética , Escherichia coli/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Mutação , Fenótipo , Plasmídeos/química , Plasmídeos/metabolismo , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/metabolismoRESUMO
BACKGROUND: The General Control Nonderepressible 2 (GCN2) kinase is a conserved member of the integrated stress response (ISR) pathway that represses protein translation and helps cells to adapt to conditions of nutrient shortage. As such, GCN2 is required for longevity and stress resistance induced by dietary restriction (DR). IMPACT is an ancient protein that inhibits GCN2. RESULTS: Here, we tested whether IMPACT down-regulation mimics the effects of DR in C. elegans. Knockdown of the C. elegans IMPACT homolog impt-1 activated the ISR pathway and increased lifespan and stress resistance of worms in a gcn-2-dependent manner. Impt-1 knockdown exacerbated DR-induced longevity and required several DR-activated transcription factors to extend lifespan, among them SKN-1 and DAF-16, which were induced during larval development and adulthood, respectively, in response to impt-1 RNAi. CONCLUSIONS: IMPACT inhibits the ISR pathway, thus limiting the activation of stress response factors that are beneficial during aging and required under DR.