RESUMO
mRNA stability is a major determinant of inflammatory gene expression. Rapid degradation of interleukin-8 (IL-8) mRNA is imposed by a bipartite AU-rich element (ARE) in the 3' untranslated region (R. Winzen et al., Mol. Cell. Biol. 24:4835-4847, 2004). Small interfering RNA-mediated knockdown of the ARE-binding protein KSRP resulted in stabilization of IL-8 mRNA or of a beta-globin reporter mRNA containing the IL-8 ARE. Rapid deadenylation was impaired, indicating a crucial role for KSRP in this step of mRNA degradation. The two IL-8 ARE domains both contribute to interaction with KSRP, corresponding to the importance of both domains for rapid degradation. Exposure to the inflammatory cytokine IL-1 has been shown to stabilize IL-8 mRNA through p38 mitogen-activated protein (MAP) kinase and MK2. IL-1 treatment impaired the interaction of KSRP with the IL-8 ARE in a manner dependent on p38 MAP kinase but apparently independent of MK2. Instead, evidence that TTP, a target of MK2, can also destabilize the IL-8 ARE reporter mRNA is presented. In a comprehensive approach to identify mRNAs controlled by KSRP, two criteria were evaluated by microarray analysis of (i) association of mRNAs with KSRP in pulldown assays and (ii) increased amounts in KSRP knockdown cells. According to both criteria, a group of 100 mRNAs is controlled by KSRP, many of which are unstable and encode proteins involved in inflammation. These results indicate that KSRP functions as a limiting factor in inflammatory gene expression.
Assuntos
Inflamação/genética , Interleucina-8/genética , Proteínas de Ligação a RNA/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Transativadores/metabolismo , Ativação Enzimática , Regulação da Expressão Gênica , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Poli A/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Tristetraprolina/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
We have previously observed rapid and strong inhibition of mRNA deadenylation and degradation in response to UV-B light [Gowrishankar et al., Biol. Chem. 386 (2005), pp. 1287-1293]. Expression analysis using a microarray for inflammatory genes showed that UV-B light induces stabilization of all short-lived mRNAs assayed. Stabilization was observed in HeLa cells, as well as in the keratinocyte line HaCaT. It affected constitutively expressed mRNA species, as well as species induced by the inflammatory cytokine IL-1. Many of the latter encode proteins involved in inflammation, suggesting that stress-induced inhibition of mRNA deadenylation contributes to changes in inflammatory gene expression. Deadenylation and degradation of tet-off-expressed mRNAs were also inhibited upon exposure to H2O2. However, scavengers of reactive oxygen species did not interfere with UV-B-induced inhibition of degradation, arguing against the involvement of UV-induced H2O2 in these effects of UV-B light. Heat shock and hyperosmolarity also inhibited mRNA deadenylation and degradation, whereas gamma-radiation did not. Thus, inhibition of mRNA deadenylation and degradation is a cellular response elicited by several but not all inducers of cell stress.
Assuntos
Adenina/metabolismo , Expressão Gênica/efeitos da radiação , Células HeLa/efeitos da radiação , Queratinócitos/efeitos da radiação , RNA Mensageiro/efeitos da radiação , Raios Ultravioleta , Citocinas/metabolismo , Eletroforese em Gel de Poliacrilamida , Células HeLa/metabolismo , Resposta ao Choque Térmico , Humanos , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Inflamação/induzido quimicamente , Interleucina-1/metabolismo , Queratinócitos/metabolismo , Concentração Osmolar , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/metabolismo , Temperatura , Fatores de TempoRESUMO
Post-transcriptional mechanisms contribute to the changes in gene expression induced by cell stress. The effect of UV-B light on mRNA degradation in HeLa cells was investigated using a transcriptional chase system to determine the decay kinetics of tet-off vector-derived mRNAs containing or lacking a destabilizing AU-rich element. Degradation of both mRNAs was strongly inhibited in cells exposed to UV-B light. Removal of the poly(A)-tail, considered a crucial step in mRNA degradation, was strikingly impaired. UV light also inhibited deadenylation and degradation of endogenous mRNA of the chemoattractant cytokine interleukin (IL)-8. Both effects occurred rapidly and independently of newly induced genes. Importantly, stabilization of IL-8 mRNA was accompanied by a strong increase in the duration of IL-8 protein formation. Furthermore, general inhibition of protein synthesis, a hallmark of the response to cell stress, required far higher doses of UV-B than inhibition of mRNA deadenylation and degradation. The difference in sensitivity of cells to these effects of UV-B light establishes a dose range in which mRNA stabilization can lead to dramatically enhanced expression of proteins derived from normally unstable mRNAs, such as those of inflammatory cytokines, growth factors and proto-oncogenes, and thereby have a major impact on the response to UV light.
Assuntos
Adenina/metabolismo , Estabilidade de RNA/efeitos da radiação , RNA Mensageiro/efeitos da radiação , Raios Ultravioleta , Adenina/antagonistas & inibidores , Citocinas/genética , Citocinas/metabolismo , Eletroforese em Gel de Poliacrilamida , Células HeLa/metabolismo , Células HeLa/efeitos da radiação , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , Cinética , Poli A/genética , Poli A/metabolismo , Estabilidade de RNA/genética , Estabilidade de RNA/fisiologia , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/metabolismoRESUMO
AU-rich elements (AREs) control the expression of numerous genes by accelerating the decay of their mRNAs. Rapid decay and deadenylation of beta-globin mRNA containing AU-rich 3' untranslated regions of the chemoattractant cytokine interleukin-8 (IL-8) are strongly attenuated by activating the p38 mitogen-activated protein (MAP) kinase/MAP kinase-activated protein kinase 2 (MK2) pathway. Further evidence for a crucial role of the poly(A) tail is provided by the loss of destabilization and kinase-induced stabilization in ARE RNAs expressed as nonadenylated forms by introducing a histone stem-loop sequence. The minimal regulatory element in the IL-8 mRNA is located in a 60-nucleotide evolutionarily conserved sequence with a structurally and functionally bipartite character: a core domain with four AUUUA motifs and limited destabilizing function on its own and an auxiliary domain that markedly enhances destabilization exerted by the core domain and thus is essential for the rapid removal of RNA targets. A similar bipartite structure and function are observed for the granulocyte-macrophage colony-stimulating factor (GM-CSF) ARE. Stabilization in response to p38/MK2 activation is seen with the core domain alone and also after mutation of the AUUUA motifs in the complete IL-8 ARE. Stabilization by ARE binding protein HuR requires different sequence elements. Binding but no stabilization is observed with the IL-8 ARE. Responsiveness to HuR is gained by exchanging the auxiliary domain of the IL-8 ARE with that of GM-CSF or with a domain of the c-fos ARE, which results in even stronger responsiveness. These results show that distinct ARE domains differ in function with regard to destabilization, stabilization by p38/MK2 activation, and stabilization by HuR.