Identification of RNA-binding proteins in RAW 264.7 cells that recognize a lipopolysaccharide-responsive element in the 3-untranslated region of the murine cyclooxygenase-2 mRNA
Identification of RNA-binding proteins in RAW 264.7 cells that recognize a lipopolysaccharide-responsive element in the 3-untranslated region of the murine cyclooxygenase-2 mRNA
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DOI：
10.1074/jbc.m308475200
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发表时间：
2004-02-27
影响因子：
4.8
通讯作者：
Morrison, AR
中科院分区：
文献类型：
--
作者：
Cok, SJ;Acton, SJ;Morrison, AR
RAW 264.7细胞响应于脂多糖处理而快速诱导环氧合酶-2（考克斯-2）。部分增加的考克斯-2表达是通过转录后机制发生的，该机制通过信使的3 '-非翻译区（UTR）的特定区域介导。考克斯-2的3 '-UTR的近端区域含有高度保守的富含AU的元件，该元件能够赋予嵌合体基因的脂多糖调节。电泳迁移率变动分析表明，RNA结合蛋白TIAR、AUF 1、HuR和TIA-1均与考克斯-2的3 '-UTR的前60个核苷酸形成RNA-蛋白复合物。生物素化的RNA探针用于分离结合考克斯-2的3 '-UTR的另外的蛋白质。我们鉴定了几种RNA结合蛋白，包括TIAR、AUF 1、CBF-A、RBM 3、异质核核糖核蛋白（hnRNP）A3和hnRNP A2/B1。我们确定了四个选择性剪接亚型的AUF 1迁移在多个等电点。同样，我们鉴定了CBF-A、hnRNP A3和hnRNP A2/B1的可变剪接亚型。二维凝胶的Western分析鉴定了TIA-1、TIAR和AUF 1的多种亚型，其pI值跨越近3个pH单位。因此，通过选择性剪接和翻译后修饰的组合，细胞能够大大增加在给定时间或响应于细胞外刺激而表达的蛋白质种类的库。
RAW 264.7 cells rapidly induce cyclooxygenase-2 (COX-2) in response to lipopolysaccharide treatment. Part of the increased COX-2 expression occurred through post-transcriptional mechanisms mediated through specific regions of the 3'-untranslated region (UTR) of the message. The proximal region of the 3'-UTR of COX-2 contains a highly conserved AU-rich element that was able to confer lipopolysaccharide regulation of a chimeric reporter-gene. Electrophoretic mobility shift assays demonstrated that the RNA-binding proteins TIAR, AUF1, HuR, and TIA-1 all form an RNA-protein complex with the first 60 nucleotides of the 3'-UTR of COX-2. Biotinylated RNA probes were used to isolate additional proteins that bind the 3'-UTR of COX-2. We identified several RNA-binding proteins including TIAR, AUF1, CBF-A, RBM3, heterogeneous nuclear ribonucleoprotein (hnRNP) A3, and hnRNP A2/B1. We identified four alternatively spliced isoforms of AUF1 which migrated at multiple isoelectric points. Likewise, we identified alternatively spliced isoforms of CBF-A, hnRNP A3, and hnRNP A2/B1. Western analysis of two-dimensional gels identified multiple isoforms of TIA-1, TIAR, and AUF1 at pI values that spanned nearly 3 pH units. Thus, through a combination of alternative splicing and post-translational modification cells are able to increase greatly the repertoire of protein species expressed at a given time or in response to extracellular stimuli.