Post-transcriptional regulation of glucose transporter-1 by an AU-rich element in the 3'UTR and by hnRNP A2.
Post-transcriptional regulation of glucose transporter-1 by an AU-rich element in the 3'UTR and by hnRNP A2.
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DOI：
10.1016/j.bbrc.2004.04.128
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发表时间：
2004-06
影响因子：
3.1
通讯作者：
M. Griffin;B. Hamilton;Kristen M Roy;M. Du;A. M. Willson;Benjamin J. Keenan;X. W. Wang;R. Nichols
中科院分区：
文献类型：
--
作者：
M. Griffin;B. Hamilton;Kristen M Roy;M. Du;A. M. Willson;Benjamin J. Keenan;X. W. Wang;R. Nichols
葡萄糖转运蛋白-1(GLUT1)介导葡萄糖的摄取，在某些癌症中表达上调。这种膜蛋白的数量受转录后机制的调节，在这种机制中，mRNA结合蛋白识别mRNA的3‘-未翻译(3’UTR)中的顺式作用元件。为了鉴定GLUT1基因中的顺式元件，我们在荧光素酶基因的3‘端非编码区构建了一个报告结构。30nt的富含腺苷尿苷元件(“GLUT1Aure”)抑制HEK-293细胞的荧光素酶活性。通过从含有全长3‘UTR的报告中删除GLUT1 Aure，证实了这种抑制作用。GLUT1 Aure基因的缺失导致报告基因活性增加。含有GLUT1的较大片段(“BSU”区)的缺失进一步增加了报告基因的活性，这表明在GLUT1 mRNA中有额外的顺式元件。GLUT1Aure在GBM-T98G胶质母细胞瘤细胞中也有活性。接下来，我们测试了反式作用因子hnRNP A2对GLUT1基因表达的作用。我们证明了胞质定位的hnRNP A2亚型通过凝胶移位实验和紫外光交联法与人GLUT1 RNA结合。最后，在GBM-T98G细胞中过表达hnRNP A2亚型抑制了GLUT1报告基因的表达。这些结果证实了人GLUT1基因中的Aure顺式元件，表明hnRNP A2作用于GLUT1基因可以抑制GLUT1基因在脑癌细胞中的表达。
Glucose transporter-1 (GLUT1) mediates uptake of glucose and is up-regulated in some cancers. The amount of this membrane protein is regulated by a post-transcriptional mechanism in which mRNA binding proteins recognize cis-acting elements in the 3′-untranslated (3′UTR) of the mRNA. To identify cis elements in GLUT1 mRNA we introduced 3′UTR sequences into the 3′UTR of the luciferase gene in a reporter construct. A 30nt adenosine–uridine-rich element (“GLUT1 AURE”) inhibited luciferase activity in HEK-293 cells. This inhibitory effect was confirmed by deleting the GLUT1 AURE from a reporter containing the full-length 3′UTR. Deletion of the GLUT1 AURE caused reporter activity to increase. Deletion of a larger fragment (“Bsu” region) containing the GLUT1 AURE increased reporter activity still further, suggesting that there are additional cis elements in the GLUT1 mRNA. The GLUT1 AURE was also active in GBM-T98G glioblastoma cells. Next, we tested the action of a trans-acting factor, hnRNP A2, on GLUT1 gene expression. We show that a cytoplasmic-localizing isoform of hnRNP A2 binds human GLUT1 RNA by gel-shift assay and by UV-crosslinking. Finally, over-expression of the hnRNP A2 isoform inhibited GLUT1 reporter expression in GBM-T98G cells. These results identify the AURE cis element in human GLUT1 mRNA and show that hnRNP A2 acts on GLUT1 mRNA to inhibit expression of GLUT1 in a brain cancer cell line.