RESUMO
Precise regulation of Notch signaling is essential for normal vertebrate development. Mind bomb (Mib) is a ubiquitin ligase that is required for activation of Notch by Notch׳s ligand, Delta. Sorting Nexin 5 (SNX5) co-localizes with Mib and Delta complexes and has been shown to directly bind to Mib. We show that microRNA-216a (miR-216a) is expressed in the retina during early development and regulates snx5 to precisely regulate Notch signaling. miR-216a and snx5 have complementary expression patterns. Knocking down miR-216a and/or overexpression of snx5 resulted in increased Notch activation. Conversely, knocking down snx5 and/or miR-216a overexpression caused a decrease in Notch activation. We propose a model in which SNX5, precisely controlled by miR-216a, is a vital partner of Mib in promoting endocytosis of Delta and subsequent activation of Notch signaling.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , MicroRNAs/metabolismo , Retina/embriologia , Transdução de Sinais/fisiologia , Nexinas de Classificação/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Análise de Variância , Animais , Clonagem Molecular , Primers do DNA/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genética , Técnicas de Silenciamento de Genes , Processamento de Imagem Assistida por Computador , Immunoblotting , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , MicroRNAs/genética , Análise em Microsséries , Modelos Biológicos , Receptores Notch/metabolismo , Retina/metabolismo , Transdução de Sinais/genéticaRESUMO
SNAP-25 is a core component of the trimeric SNARE complex mediating vesicle exocytosis during membrane addition for neuronal growth, neuropeptide/growth factor secretion, and neurotransmitter release during synaptic transmission. Here, we report a novel microRNA mechanism of SNAP-25 regulation controlling motor neuron development, neurosecretion, synaptic activity, and movement in zebrafish. Loss of miR-153 causes overexpression of SNAP-25 and consequent hyperactive movement in early zebrafish embryos. Conversely, overexpression of miR-153 causes SNAP-25 down regulation resulting in near complete paralysis, mimicking the effects of treatment with Botulinum neurotoxin. miR-153-dependent changes in synaptic activity at the neuromuscular junction are consistent with the observed movement defects. Underlying the movement defects, perturbation of miR-153 function causes dramatic developmental changes in motor neuron patterning and branching. Together, our results indicate that precise control of SNAP-25 expression by miR-153 is critically important for proper neuronal patterning as well as neurotransmission.
Assuntos
MicroRNAs/fisiologia , Neurônios Motores/citologia , Transmissão Sináptica/fisiologia , Proteína 25 Associada a Sinaptossoma/fisiologia , Animais , Sequência de Bases , Exocitose/fisiologia , Proteínas de Fluorescência Verde/genética , MicroRNAs/genética , Homologia de Sequência de Aminoácidos , Transdução de Sinais/fisiologia , Peixe-Zebra/embriologiaRESUMO
microRNAs (miRNAs) are a family of 21-23 nucleotide endogenous non-coding RNAs that post-transcriptionally regulate gene expression in a sequence-specific manner. Typically, miRNAs downregulate target genes by recognizing and recruiting protein complexes to 3'UTRs, followed by translation repression or mRNA degradation. miR-92 is a well-studied oncogene in mammalian systems. Here, using zebrafish as a model system, we uncovered a novel tissue-inductive role for miR-92 during early vertebrate development. Overexpression resulted in reduced endoderm formation during gastrulation with consequent cardia and viscera bifida. By contrast, depletion of miR-92 increased endoderm formation, which led to abnormal Kupffer's vesicle development and left-right patterning defects. Using target prediction algorithms and reporter constructs, we show that gata5 is a target of miR-92. Alteration of gata5 levels reciprocally mirrored the effects of gain and loss of function of miR-92. Moreover, genetic epistasis experiments showed that miR-92-mediated defects could be substantially suppressed by modulating gata5 levels. We propose that miR-92 is a critical regulator of endoderm formation and left-right asymmetry during early zebrafish development and provide the first evidence for a regulatory function for gata5 in the formation of Kupffer's vesicle and left-right patterning.
Assuntos
Padronização Corporal/genética , Endoderma/embriologia , MicroRNAs/fisiologia , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados , Sequência de Bases , Cárdia/embriologia , Cárdia/metabolismo , Embrião não Mamífero , Endoderma/metabolismo , Fator de Transcrição GATA5/genética , Fator de Transcrição GATA5/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/genética , MicroRNAs/metabolismo , Distribuição Tecidual , Vísceras/embriologia , Vísceras/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
microRNAs (miRNAs) are small ( approximately 22 nt) noncoding RNAs that have been shown to regulate gene expression post-transcriptionally. They function by pairing with the 3' UTR of target mRNAs and repressing translation or by targeting the mRNA for degradation. miRNAs are involved in diverse aspects of development, maintenance, and disease, and are largely evolutionarily conserved in metazoans. Searching the genomes of organisms from viruses to worms to humans has revealed potentially thousands of miRNA genes. Understanding the patterns of genomic organization between species cannot only help to refine tools to identify new miRNAs, but also provide insight into miRNA biogenesis and function.
