RESUMO
Injury response in the peripheral nervous system (PNS) is characterized by rapid alterations in the genetic program of Schwann cells. However, the epigenetic mechanisms modulating these changes remain elusive. Here we show that sciatic nerve injury in mice induces a cohort of 22 miRNAs, which coordinate Schwann cell differentiation and dedifferentiation through a combinatorial modulation of their positive and negative gene regulators. These miRNAs and their targeted mRNAs form functional complexes with the Argonaute-2 protein to mediate post-transcriptional gene silencing. MiR-138 and miR-709 show the highest affinity amongst the cohort, for binding and regulation of Egr2, Sox-2 and c-Jun expression following injury. Moreover, miR-709 participates in the formation of epigenetic silencing complexes with H3K27me3 and Argonaute-1 to induce transcriptional gene silencing of the Egr2 promoter. Collectively, we identified a discrete cohort of miRNAs as the central epigenetic regulators of the transition between differentiation and dedifferentiation during the acute phase of PNS injury.
Assuntos
Regulação da Expressão Gênica , MicroRNAs/genética , Traumatismos dos Nervos Periféricos/genética , Interferência de RNA , Transcrição Gênica , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Diferenciação Celular/genética , Proteína 2 de Resposta de Crescimento Precoce/genética , Feminino , Perfilação da Expressão Gênica , Ordem dos Genes , Camundongos , MicroRNAs/metabolismo , Traumatismos dos Nervos Periféricos/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Fatores de Transcrição SOXB1/genética , Células de Schwann/citologia , Células de Schwann/metabolismo , Nervo Isquiático/lesõesRESUMO
Reciprocal interactions between glia and neurons are essential for the proper organization and function of the nervous system. Recently, the interaction between ErbB receptors (ErbB2 and ErbB3) on the surface of Schwann cells and neuronal Neuregulin-1 (NRG1) has emerged as the pivotal signal that controls Schwann cell development, association with axons, and myelination. To understand the function of NRG1-ErbB2/3 signaling axis in adult Schwann cell biology, we are studying the specific role of ErbB3 receptor tyrosine kinase (RTK) since it is the receptor for NRG1 on the surface of Schwann cells. Here, we show that alternative transcription initiation results in the formation of a nuclear variant of ErbB3 (nuc-ErbB3) in rat primary Schwann cells. nuc-ErbB3 possesses a functional nuclear localization signal sequence and binds to chromatin. Using chromatin immunoprecipitation (ChIP)-chip arrays, we identified the promoters that associate with nuc-ErbB3 and clustered the active promoters in Schwann cell gene expression. nuc-ErbB3 regulates the transcriptional activity of ezrin and HMGB1 promoters, whereas inhibition of nuc-ErbB3 expression results in reduced myelination and altered distribution of ezrin in the nodes of Ranvier. Finally, we reveal that NRG1 regulates the translation of nuc-ErbB3 in rat Schwann cells. For the first time, to our knowledge, we show that alternative transcription initiation from a gene that encodes a RTK is capable to generate a protein variant of the receptor with a distinct role in molecular and cellular regulation. We propose a new concept for the molecular regulation of myelination through the expression and distinct role of nuc-ErbB3.
Assuntos
Núcleo Celular/enzimologia , Proteínas do Citoesqueleto/metabolismo , Variação Genética/fisiologia , Bainha de Mielina/enzimologia , Receptor ErbB-3/fisiologia , Células de Schwann/enzimologia , Processamento Alternativo/genética , Animais , Animais Recém-Nascidos , Células COS , Núcleo Celular/genética , Chlorocebus aethiops , Técnicas de Cocultura , Proteínas do Citoesqueleto/genética , Feminino , Masculino , Bainha de Mielina/química , Bainha de Mielina/genética , Ratos , Receptor ErbB-3/genética , Células de Schwann/químicaRESUMO
We used a high flux synchrotron X-ray beam to map the structure of 16S rRNA and RNase P in viable bacteria in situ. A 300 ms exposure to the X-ray beam was sufficient for optimal cleavage of the phosphodiester backbone. The in vivo footprints of the 16S rRNA in frozen cells were similar to those obtained in vitro and were consistent with the predicted accessibility of the RNA backbone to hydroxyl radical. Protection or enhanced cleavage of certain nucleotides in vivo can be explained by interactions with tRNA and perturbation of the subunit interface. Thus, short exposures to a synchrotron X-ray beam can footprint the tertiary structure and protein contacts of RNA-protein complexes with nucleotide resolution in living cells.
Assuntos
Radical Hidroxila , RNA/química , Síncrotrons , Sequência de Bases , Escherichia coli/genética , Escherichia coli/efeitos da radiação , Técnicas Genéticas/instrumentação , Dados de Sequência Molecular , RNA/metabolismo , RNA/efeitos da radiação , RNA Ribossômico 16S/química , Ribonuclease P/química , Raios XRESUMO
Coordinate regulation of the ribosomal protein genes is entrusted to a number of signal transduction pathways that can abruptly induce or silence the ribosomal genes. We have uncovered a cellular model system, which selectively induces the ribosomal protein S25 gene in hepatoma cells that are stressed by nutrient deprivation. Our results indicate that p53 along with two other identified proteins, MTF-1 and La, post-transcriptionally regulate the synthesis of the S25 protein by controlling the nuclear export of the stress-induced S25 mRNA. This system is unique in that the nuclear-retained S25 mRNA is exported to the cytosol only upon replenishment or alternatively after prolonged starvation to participate in a p53-mediated apoptotic sequence of events. This p53-dependent survival or death pathway involves a previously unreported protein relationship among these three actors, one of which, MTF-1, has not yet been shown to have RNA-binding characteristics.