RESUMO
Circular RNAs (circRNAs) are single-stranded RNAs that are joined head to tail with largely unknown functions. Here we show that transfection of purified in vitro generated circRNA into mammalian cells led to potent induction of innate immunity genes and confers protection against viral infection. The nucleic acid sensor RIG-I is necessary to sense foreign circRNA, and RIG-I and foreign circRNA co-aggregate in cytoplasmic foci. CircRNA activation of innate immunity is independent of a 5' triphosphate, double-stranded RNA structure, or the primary sequence of the foreign circRNA. Instead, self-nonself discrimination depends on the intron that programs the circRNA. Use of a human intron to express a foreign circRNA sequence abrogates immune activation, and mature human circRNA is associated with diverse RNA binding proteins reflecting its endogenous splicing and biogenesis. These results reveal innate immune sensing of circRNA and highlight introns-the predominant output of mammalian transcription-as arbiters of self-nonself identity.
Assuntos
Vírus da Encefalite Equina Venezuelana/imunologia , Encefalomielite Equina Venezuelana/prevenção & controle , Tolerância Imunológica , Imunidade Inata , Íntrons , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/imunologia , RNA/genética , RNA/imunologia , Animais , Sequência de Bases , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/imunologia , Proteína DEAD-box 58/metabolismo , Vírus da Encefalite Equina Venezuelana/genética , Vírus da Encefalite Equina Venezuelana/metabolismo , Encefalomielite Equina Venezuelana/genética , Encefalomielite Equina Venezuelana/imunologia , Encefalomielite Equina Venezuelana/metabolismo , Células HEK293 , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Tolerância Imunológica/genética , Imunidade Inata/genética , Camundongos , Conformação de Ácido Nucleico , Ligação Proteica , Células RAW 264.7 , RNA/biossíntese , RNA/química , RNA Circular , RNA Mensageiro/genética , RNA Mensageiro/imunologia , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Receptores Imunológicos , Spliceossomos/imunologia , Spliceossomos/metabolismo , TransfecçãoRESUMO
Peroxisomes cooperate with mitochondria in the performance of cellular metabolic functions, such as fatty acid oxidation and the maintenance of redox homeostasis. However, whether peroxisomes also regulate mitochondrial fission-fusion dynamics or mitochondrion-dependent apoptosis remained unclear. We now show that genetic ablation of the peroxins Pex3 or Pex5, which are essential for peroxisome biogenesis, results in mitochondrial fragmentation in mouse embryonic fibroblasts (MEFs) in a manner dependent on Drp1 (also known as DNM1L). Conversely, treatment with 4-PBA, which results in peroxisome proliferation, resulted in mitochondrial elongation in wild-type MEFs, but not in Pex3-knockout MEFs. We further found that peroxisome deficiency increased the levels of cytosolic cytochrome c and caspase activity under basal conditions without inducing apoptosis. It also greatly enhanced etoposide-induced caspase activation and apoptosis, which is indicative of an enhanced cellular sensitivity to death signals. Taken together, our data unveil a previously unrecognized role for peroxisomes in the regulation of mitochondrial dynamics and mitochondrion-dependent apoptosis. Effects of peroxin gene mutations on mitochondrion-dependent apoptosis may contribute to pathogenesis of peroxisome biogenesis disorders.This article has an associated First Person interview with the first author of the paper.
Assuntos
Apoptose/fisiologia , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/fisiologia , Peroxissomos/metabolismo , Animais , Butilaminas/farmacologia , Caspases/metabolismo , Linhagem Celular , Citocromos c/metabolismo , Dinaminas/metabolismo , Humanos , Lipoproteínas/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Peroxinas/genética , Transtornos Peroxissômicos/patologia , Receptor 1 de Sinal de Orientação para Peroxissomos/genética , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
A "cytokine-less" in vitro differentiation method would be promising for cost-effective mass production of cells used for regenerative medicine. In this study, we developed a differentiation signalobody S-RANK, in which the extracellular domain of receptor activator of nuclear factor kappa-B (RANK) is replaced with a single-chain variable fragment (scFv) to attain signaling in response to an inexpensive antigen. A murine macrophage cell line RAW264, which is known to differentiate into an osteoclast by RANK ligand (RANKL), was lentivirally transduced with S-RANK. When the resultant cells were cultured with a specific antigen, the cells differentiated into multinucleated tartrate-resistant acid phosphatase-positive osteoclasts. The differentiation efficiency was almost comparable to those induced by RANKL. In addition, the signaling analysis demonstrated that nuclear factor kappa-B and mitogen-activated protein kinase signaling pathways, which are the major signaling pathways downstream of wild-type RANK, were also activated by S-RANK. These results demonstrate that S-RANK sufficiently mimics signal transduction of wild-type RANK. Differentiation signalobodies may be applied for controlling differentiation of other cell types by using appropriate signaling domains.