Natural antisense transcript stabilizes inducible nitric oxide synthase messenger RNA in rat hepatocytes
Natural antisense transcript stabilizes inducible nitric oxide synthase messenger RNA in rat hepatocytes
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DOI：
10.1002/hep.22036
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发表时间：
2008-02-01
期刊：
影响因子：
13.5
通讯作者：
Okumura, Tadayoshi
中科院分区：
文献类型：
--
作者：
Matsui, Kosuke;Nishizawa, Mikio;Okumura, Tadayoshi
在炎症过程中，诱导型一氧化氮合酶(INOS)被诱导产生重要的介质一氧化氮(NO)。白介素1β(IL-1β)诱导大鼠肝细胞iNOS信使RNA(MRNA)、iNOS蛋白和NO的表达。我们发现诱导型一氧化氮合酶基因的稳定性在诱导过程中发生了变化，与诱导型一氧化氮合酶基因3‘非翻译区相对应的反义(AS)链从诱导型一氧化氮合酶基因转录而来。INOS AS转录本的表达水平与iNOS mRNA的表达水平相关。INOS基因外显子27的3‘侧翼1.5kb碱基区参与了IL-1β的诱导。基因敲除实验表明，针对iNOS mRNA的正义寡核苷酸通过阻断iNOS mRNA与AS转录本之间的相互作用，显著降低了肝细胞中iNOS mRNA的水平。INOS作为转录产物的过表达通过融合的iNOS mRNA3‘非编码区稳定了报告荧光素酶的mRNA。这些结果与酵母RNA杂交实验结果相结合，表明iNOS作为转录本与iNOS mRNA相互作用，并稳定了iNOS mRNA。在大鼠肝细胞胞浆中，NOS mRNA与人类胚胎致死异常视觉蛋白的同源物--富含AU的元件结合蛋白HUR和异质性核糖核蛋白L(HnRNP L)共存。相互作用分析进一步表明，iNOS AS转录本与HUR相互作用，而HUR又与hnRNP L相互作用，提示iNOS mRNA、AS转录本和RNA结合蛋白可能相互作用。结论：iNOS基因的天然AS转录本与iNOS mRNA相互作用，可能在iNOS mRNA的稳定性中起重要作用。这种RNA-RNA相互作用可能成为治疗非介导性炎症性疾病的新靶点。
During inflammation, inducible nitric oxide synthase (iNOS) is induced to generate the important mediator nitric oxide (NO). Interleukin 1 beta (IL-1 beta) induces iNOS messenger RNA (mRNA), iNOS protein, and NO in rat hepatocytes. We found that the stability of iNOS mRNA changed during the induction and that the antisense (AS) strand corresponding to the 3'-untranslated region (3'UTR) of iNOS mRNA was transcribed from the iNOS gene. Expression levels of the iNOS AS transcript correlated with those of iNOS mRNA. The 1.5-kilobase region 3'-flanking to iNOS gene exon 27 was involved in IL-1 beta induction. Knockdown experiments suggest that sense oligonucleotides to iNOS mRNA significantly reduced iNOS mRNA levels in the hepatocytes by blocking the interaction between iNOS mRNA and the AS transcript. Overexpression of iNOS AS transcript stabilized the reporter luciferase mRNA through the fused iNOS mRNA 3'UTR. These results together with the data in a yeast RNA-hybrid assay suggested that the iNOS AS transcript interacted with iNOS mRNA and stabilized iNOS mRNA. The NOS mRNA colocalized with the AU-rich element-binding protein HuR, a human homolog of embryonic lethal-abnormal visual protein, and heterogeneous nuclear ribonucleoprotein L (hnRNP L) in the cytoplasm of rat hepatocytes. Interaction assays further revealed that the iNOS AS transcript interacted with HuR, which interacted with hnRNP L, suggesting that iNOS mRNA, the AS transcript, and the RNA-binding proteins may mutually interact. Conclusion: The natural AS transcript of the iNOS gene interacts with iNOS mRNA and may play an important role in the stability of iNOS mRNA. This RNA-RNA interaction may be a new therapeutic target for NO-mediating inflammatory diseases.