RESUMO
MicroRNAs (miRNAs) are genome-encoded small double-stranded RNAs that have emerged as key regulators of gene expression and are implicated in most aspects of human development and disease. Canonical miRNA biogenesis involves processing of â¼70-nucleotide pre-miRNA hairpins by Dicer to generate mature â¼22-nucleotide miRNAs, which target complementary RNA sequences. Despite the importance of miRNA biogenesis, signaling mechanisms controlling this process are poorly defined. Here we demonstrate that the post-transcriptional regulation of Dicer is controlled by the cell density-mediated localization of the Hippo pathway effectors TAZ (transcriptional co-activator with PDZ-binding motif) and YAP (Yes-associated protein) (TAZ/YAP). We show that nuclear TAZ/YAP, which are abundant at low cell density, are required for efficient pre-miRNA processing. Knockdown of TAZ/YAP in low density cells, or density-mediated sequestration of TAZ/YAP into the cytoplasm, results in the defective processing of pre-miRNAs. Strikingly, one exception is Let-7, which accumulates upon loss of nuclear TAZ/YAP, leading to Let-7-dependent reduction in Dicer levels. Accordingly, inhibition of Let-7 rescues the miRNA biogenesis defects observed following TAZ/YAP knockdown. Thus, density-regulated TAZ/YAP localization defines a critical and previously unrecognized mechanism by which cells relay cell contact-induced cues to control miRNA biogenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , RNA Helicases DEAD-box/biossíntese , Regulação Enzimológica da Expressão Gênica/fisiologia , MicroRNAs/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ribonuclease III/biossíntese , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Aciltransferases , Proteínas Adaptadoras de Transdução de Sinal/genética , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , RNA Helicases DEAD-box/genética , Técnicas de Silenciamento de Genes , Via de Sinalização Hippo , Humanos , MicroRNAs/genética , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinases/genética , Ribonuclease III/genética , Fatores de Transcrição/genética , Proteínas de Sinalização YAPRESUMO
Hemojuvelin (Hjv), a member of the repulsive-guidance molecule (RGM) family, upregulates transcription of the iron regulatory hormone hepcidin by activating the bone morphogenetic protein (BMP) signaling pathway in mammalian cells. Mammalian models have identified furin, neogenin, and matriptase-2 as modifiers of Hjv's function. Using the zebrafish model, we evaluated the effects of hjv and its interacting proteins on hepcidin expression during embryonic development. We found that hjv is strongly expressed in the notochord and somites of the zebrafish embryo and that morpholino knockdown of hjv impaired the development of these structures. Knockdown of hjv or other hjv-related genes, including zebrafish orthologs of furin or neogenin, however, failed to decrease hepcidin expression relative to liver size. In contrast, overexpression of bmp2b or knockdown of matriptase-2 enhanced the intensity and extent of hepcidin expression in zebrafish embryos, but this occurred in an hjv-independent manner. Furthermore, we demonstrated that zebrafish hjv can activate the human hepcidin promoter and enhance BMP responsive gene expression in vitro, but is expressed at low levels in the zebrafish embryonic liver. Taken together, these data support an alternative mechanism for hepcidin regulation during zebrafish embryonic development, which is independent of hjv.
Assuntos
Peptídeos Catiônicos Antimicrobianos/análise , Proteínas Morfogenéticas Ósseas/metabolismo , Transdução de Sinais , Transativadores/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/metabolismo , Animais , Antibacterianos , Peptídeos Catiônicos Antimicrobianos/biossíntese , Peptídeos Catiônicos Antimicrobianos/genética , Proteína Morfogenética Óssea 2 , Embrião não Mamífero , Proteínas Ligadas por GPI , Hemocromatose/congênito , Proteína da Hemocromatose , Hepcidinas , Humanos , Fígado/química , Fígado/metabolismo , Notocorda/química , Regiões Promotoras Genéticas , Serina Endopeptidases , Somitos/química , Peixe-Zebra/genéticaRESUMO
The iron regulatory hormone hepcidin is transcriptionally up-regulated in response to iron loading, but the mechanisms by which iron levels are sensed are not well understood. Large-scale genetic screens in the zebrafish have resulted in the identification of hypochromic anemia mutants with a range of mutations affecting conserved pathways in iron metabolism and heme synthesis. We hypothesized that transferrin plays a critical role both in iron transport and in regulating hepcidin expression in zebrafish embryos. Here we report the identification and characterization of the zebrafish hypochromic anemia mutant, gavi, which exhibits transferrin deficiency due to mutations in transferrin-a. Morpholino knockdown of transferrin-a in wild-type embryos reproduced the anemia phenotype and decreased somite and terminal gut iron staining, while coinjection of transferrin-a cRNA partially restored these defects. Embryos with transferrin-a or transferrin receptor 2 (TfR2) deficiency exhibited low levels of hepcidin expression, however anemia, in the absence of a defect in the transferrin pathway, failed to impair hepcidin expression. These data indicate that transferrin-a transports iron and that hepcidin expression is regulated by a transferrin-a-dependent pathway in the zebrafish embryo.
