RESUMO
Mutations in RanBP2 (also known as Nup358), one of the main components of the cytoplasmic filaments of the nuclear pore complex, contribute to the overproduction of acute necrotizing encephalopathy (ANE1)-associated cytokines. Here we report that RanBP2 represses the translation of the interleukin 6 (IL6) mRNA, which encodes a cytokine that is aberrantly up-regulated in ANE1. Our data indicates that soon after its production, the IL6 messenger ribonucleoprotein (mRNP) recruits Argonautes bound to let-7 microRNA. After this mRNP is exported to the cytosol, RanBP2 sumoylates mRNP-associated Argonautes, thereby stabilizing them and enforcing mRNA silencing. Collectively, these results support a model whereby RanBP2 promotes an mRNP remodelling event that is critical for the miRNA-mediated suppression of clinically relevant mRNAs, such as IL6.
Assuntos
Proteínas Argonautas/genética , Fatores de Iniciação em Eucariotos/genética , Regulação da Expressão Gênica , MicroRNAs/genética , Chaperonas Moleculares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Regiões 3' não Traduzidas/genética , Proteínas Argonautas/metabolismo , Linhagem Celular Tumoral , Fatores de Iniciação em Eucariotos/metabolismo , Células HEK293 , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , MicroRNAs/metabolismo , Chaperonas Moleculares/metabolismo , Mutação , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Pancreatite Necrosante Aguda/genética , Pancreatite Necrosante Aguda/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , SumoilaçãoRESUMO
In eukaryotes, most mRNAs that encode secretory or membrane-bound proteins are translated by ribosomes associated with the surface of the endoplasmic reticulum (ER). Other such mRNAs are tethered to the ER by mRNA receptors. However, there has been much debate as to whether all mRNAs, regardless of their encoded polypeptide, are anchored to the ER at some low level. Here we describe a protocol to visualize ER-associated mRNAs in tissue culture cells by single-molecule fluorescence in situ hybridization (smFISH). Using this protocol, we have established that a subset of all mRNAs, regardless of whether they encode secretory or cytosolic proteins, are ER associated in a ribosome-dependent manner.