The human glucocorticoid receptor as an RNA-binding protein: global analysis of glucocorticoid receptor-associated transcripts and identification of a target RNA motif.
The human glucocorticoid receptor as an RNA-binding protein: global analysis of glucocorticoid receptor-associated transcripts and identification of a target RNA motif.
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DOI：
10.4049/jimmunol.1001794
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发表时间：
2011-01-15
期刊：
影响因子：
--
通讯作者：
Stellato C
中科院分区：
文献类型：
--
作者：
Ishmael FT;Fang X;Houser KR;Pearce K;Abdelmohsen K;Zhan M;Gorospe M;Stellato C
转录后调控正在成为糖皮质激素（GC）介导的基因调控的关键因素。我们研究了人糖皮质激素受体 (GR) 作为 RNA 结合蛋白的作用及其对人气道上皮细胞 mRNA 周转的影响。使用强效 GC 布地奈德进行细胞处理可加速 BEAS-2B 细胞中 CCL2 mRNA（t1/2=8±1 分钟 vs. DMSO 处理细胞中的 62±17 分钟）和 CCL7 mRNA（t1/2=15±4 分钟 vs. 114±37 分钟）的衰减，但不会加速 CCL5 mRNA 的衰减（t1/2=231±8 分钟 vs. 266±5 分钟）线。这种效应被抗 GR 抗体预孵育所抑制，表明 GR 本身在这些转录本的周转中发挥着作用。免疫共沉淀和生物素下拉实验表明，GR 与 CCL2 和 CCL7 mRNA 相关，但与 CCL5 mRNA 无关。这些方法证实了 GR 在人原代气道上皮细胞中靶向 CCL2 mRNA。 GR 的关联定位于 CCL2 mRNA 的 5’UTR，并进一步定位于核苷酸 44-60。通过 GR-mRNA 复合物的免疫沉淀和微阵列分析来鉴定与 GR 相关的转录本集合，揭示了 479 个与 GR 相关的转录本。对这些转录物的一级序列和二级结构的计算分析产生了富含 GC 的基序，该基序被证明在体外与 GR 结合。该基序用于预测 GR 与另外 7889 个转录物的结合。这些结果表明，细胞质 GR 通过特定序列与 mRNA 子集相互作用，并可以调节周转率，表明 GR 作为 RNA 结合蛋白具有新的转录后作用。
Posttranscriptional regulation is emerging as a key factor in glucocorticoid (GC)-mediated gene regulation. We investigated the role of the human glucocorticoid receptor (GR) as an RNA-binding protein and its effect on mRNA turnover in human airway epithelial cells. Cell treatment with the potent GC budesonide accelerated the decay of CCL2 mRNA (t1/2=8±1 min vs. 62±17 min in DMSO-treated cells) and CCL7 mRNA (t1/2=15±4 min vs. 114±37 min), but not that of CCL5 mRNA (t1/2=231±8 min vs. 266±5 min) in the BEAS-2B cell line. This effect was inhibited by pre-incubation with an anti-GR antibody, indicating that GR itself plays a role in the turnover of these transcripts. Co-immunoprecipitation and biotin pulldown experiments showed that GR associates with CCL2 and CCL7 mRNAs, but not CCL5 mRNA. These methods confirmed CCL2 mRNA targeting by GR in human primary airway epithelial cells. Association of the GR was localized to the 5’UTR of CCL2 mRNA, and further mapped to nucleotides 44–60. The collection of transcripts associated with GR, identified by immunoprecipitation of GR-mRNA complexes followed by microarray analysis, revealed 479 transcripts that associated with GR. Computational analysis of the primary sequence and secondary structures of these transcripts yielded a GC-rich motif, which was shown to bind to GR in vitro. This motif was used to predict binding of GR to an additional 7889 transcripts. These results indicate that cytoplasmic GR interacts with a subset of mRNA through specific sequences and can regulate turnover rates, suggesting a novel posttranscriptional role for GR as an RNA-binding protein.
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