Global and specific translational control by rapamycin in T cells uncovered by microarrays and proteomics
Global and specific translational control by rapamycin in T cells uncovered by microarrays and proteomics
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DOI：
10.1074/jbc.m202014200
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发表时间：
2002-06-21
影响因子：
4.8
通讯作者：
Beretta, L
中科院分区：
文献类型：
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作者：
Grolleau, A;Bowman, J;Beretta, L
雷帕霉素已被证明会影响翻译。我们利用两种互补的方法来鉴定在Jurkat T细胞中主要受雷帕霉素影响的基因。一种是使用与6300个人类基因互补的寡核苷酸微阵列来比较多聚核糖体结合的RNA和总RNA的水平。另一种是使用二维聚丙烯酰胺凝胶电泳来确定蛋白质合成水平。对多聚核糖体结合的RNA水平的表达变化的分析表明，在雷帕霉素处理后，大多数表达基因的翻译部分减少。然而，136个基因（占表达基因的6%）的翻译被完全抑制。这一组包括编码RNA结合蛋白的基因和几个蛋白酶体亚基成员。一组159个基因（占7%）的翻译在很大程度上不受雷帕霉素处理的影响。这些基因包括转录因子、激酶、磷酸酶和RAS超家族成员。使用二维聚丙烯酰胺凝胶电泳对来自相同细胞群体的[S - 35]甲硫氨酸标记的蛋白质进行分析表明，在雷帕霉素处理的细胞中，830个蛋白质斑点中的111个的综合强度变化了至少3倍（70个增加，41个减少）。我们鉴定了22个受影响的蛋白质斑点，它们代表16个基因的蛋白质产物。微阵列和蛋白质组学相结合的方法揭示了受雷帕霉素影响的新基因，这些基因可能与其免疫抑制作用有关，以及在帽依赖翻译普遍受抑制的情况下在翻译水平不受影响的其他基因。
Rapamycin has been shown to affect translation. We have utilized two complementary approaches to identify genes that are predominantly affected by rapamycin in Jurkat T cells. One was to compare levels of polysome-bound and total RNA using oligonucleotide microarrays complementary to 6,300 human genes. Another was to determine protein synthesis levels using two-dimensional PAGE. Analysis of expression changes at the polysome-bound RNA levels showed that translation of most of the expressed genes was partially reduced following rapamycin treatment. However, translation of 136 genes (6% of the expressed genes) was totally inhibited. This group included genes encoding RNA-binding proteins and several proteasome subunit members. Translation of a set of 159 genes (7%) was largely unaffected by rapamycin treatment. These genes included transcription factors, kinases, phosphatases, and members of the RAS superfamily. Analysis of [S-35]methionine-labeled proteins from the same cell populations using two-dimensional PAGE showed that the integrated intensity of 111 of 830 protein spots changed in rapamycin-treated cells by at least 3-fold (70 increased, 41 decreased). We identified 22 affected protein spots representing protein products of 16 genes. The combined microarray and proteomic approach has uncovered novel genes affected by rapamycin that may be involved in its immunosuppressive effect and other genes that are not affected at the level of translation in a context of general inhibition of cap-dependent translation.