The 3′-untranslated region of p21WAF1 mRNA is a composite cis-acting sequence bound by RNA-binding proteins from breast cancer cells, including HuR and poly(C)-binding protein
The 3′-untranslated region of p21WAF1 mRNA is a composite cis-acting sequence bound by RNA-binding proteins from breast cancer cells, including HuR and poly(C)-binding protein
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DOI：
10.1074/jbc.m208439200
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发表时间：
2003-01-31
影响因子：
4.8
通讯作者：
Leedman, PJ
中科院分区：
文献类型：
--
作者：
Giles, KM;Daly, JM;Leedman, PJ
尽管促进许多细胞类型的生长，但表皮生长因子（EGF）在过表达其受体的各种癌细胞中诱导生长抑制。细胞周期蛋白依赖性激酶抑制剂p21（WAF 1）是这一途径的核心组成部分。我们在人MDA-468乳腺癌细胞中发现，EGF通过增加mRNA稳定性和转录的组合上调p21（WAF 1）mRNA和蛋白。杂合荧光素酶报告基因全长p21（WAF 1）3 '-非翻译区（UTR）mRNA的衰减速率显著快于对照mRNA的衰减速率。用多种p21（WAF 1）3 '-UTR构建体转染鉴定了能够降低基础报告子活性的多个顺式作用元件。短波长紫外光在含有p21（WAF 1）3 '-UTR的5'区的构建体中诱导报告子活性，而EGF在含有UV-响应区的3'序列的构建体中诱导报告子活性。这些顺式元件结合来自MDA-468细胞的多种蛋白质，包括HuR和聚（C）结合蛋白1（CP 1）。免疫沉淀实验证实，在MDA-468细胞中，HuR和CP 1与p21（WAF 1）mRNA相关。HuR在MDA-468细胞中的过表达和低表达不影响EGF诱导的p21（WAF 1）蛋白表达或生长抑制。然而，HuR与其靶3 '-UTR顺式元件的结合受UVC而非EGF调节，表明这些刺激通过不同的机制调节p21（WAF 1）mRNA的稳定性。我们的结论是，EGF诱导的p21（WAF 1）蛋白的表达主要是通过稳定的p21（WAF 1）mRNA诱导通过多个3 '-UTR顺式元件介导的。虽然HuR结合这些元件中的至少一个，但它似乎不是该系统中p21（WAF 1）表达或生长抑制的主要调节剂。CP 1是一种新的p21（WAF 1）mRNA结合蛋白，可能与其他mRNA结合蛋白协同调节p21（WAF 1）mRNA的稳定性。
Despite promoting growth in many cell types, epidermal growth factor (EGF) induces growth inhibition in a variety of cancer cells that overexpress its receptor. The cyclin-dependent kinase inhibitor p21(WAF1) is a central component of this pathway. We found in human MDA-468 breast cancer cells that EGF up-regulates p21(WAF1) mRNA and protein, through a combination of increased mRNA stability and transcription. The decay rate of a hybrid luciferase reporter full-length p21(WAF1) 3'-untranslated region (UTR) mRNA was significantly faster than that of a control mRNA. Transfections with a variety of p21(WAF1) 3'-UTR constructs identified multiple cis-acting elements capable of reducing basal reporter activity. Short wavelength ultraviolet light induced reporter activity in constructs containing the 5' region of the p21(WAF1) 3'-UTR, whereas EGF induced reporter activity in constructs containing sequences 3' of the UVC-responsive region. These cis-elements bound multiple proteins from MDA-468 cells, including HuR and poly(C)-binding protein 1 (CP1). Immunoprecipitation studies confirmed that HuR and CP1 associate with p21(WAF1) mRNA in MDA-468 cells. Over- and underexpression of HuR in MDA-468 cells did not affect EGF-induced p21(WAF1) protein expression or growth inhibition. However, binding of HuR to its target 3'-UTR cis-element was regulated by UVC but not by EGF, suggesting that these stimuli modulate the stability of p21(WAF1) mRNA via different mechanisms. We conclude that EGF-induced p21(WAF1) protein expression is mediated largely by stabilization of p21(WAF1) mRNA elicited via multiple 3'-UTR cis-elements. Although HuR binds at least one of these elements, it does not appear to be a major modulator of p21(WAF1) expression or growth inhibition in this system. CP1 is a novel p21(WAF1) mRNA-binding protein that may function cooperatively with other mRNA-binding proteins to regulate p21(WAF1) mRNA stability.