Transcription profiling of mouse Gcn2 wild-type and knockout liver perfused with or without methionine

In eukaryotes, regulation of mRNA translation enables a fast, localized and finely tuned expression of gene products. Within the translation process, the first stage of translation initiation is most rigorously modulated by the actions of eukaryotic initiation factors (eIFs) and their associated proteins. These 11 eIFs catalyze the joining of the tRNA, mRNA and rRNA into a functional translation complex. Their activity is influenced by a wide variety of extra- and intracellular signals, ranging from global, such as hormone signaling and unfolded proteins, to specific, such as single amino acid imbalance and iron deficiency. Their action is correspondingly comprehensive, in increasing or decreasing recruitment and translation of most cellular mRNAs, and specialized, in targeting translation of mRNAs with regulatory features such as a 5' terminal oligopyrimidine tract (TOP), upstream open reading frames (uORFs), or an internal ribosomal entry site (IRES). In mammals, two major pathways are linked to targeted mRNA translation. The target of rapamycin (TOR) kinase induces translation of TOP and perhaps other subsets of mRNAs, whereas a family of eIF2 kinases does so with mRNAs containing uORFs or an IRES. TOR targets translation of mRNAs that code for proteins involved in translation, an action compatible with its widely accepted role in regulating cellular growth. The four members of the eIF2 kinase family increase translation of mRNAs coding for stress response proteins such as transcription factors and chaperones. Though all four kinases act on one main substrate, eIF2, published literature demonstrates both common and unique effects by each kinase in response to its specific activating stress. This suggests that the activated eIF2 kinases regulate the translation of both a global and a specific set of mRNAs. Up to now, few studies have attempted to test such a hypothesis; none has been done in mammals. We use array analysis to determine the global mRNA shift into polysomes following a stress response, and to compare the translational response following activation of GCN2 versus PERK, two of the four eIF2alpha kinases. Experiment Overall Design: Gcn2 wild-type or knockout mouse liver were perfused with complete amino acids media or media lacking methionine for RNA extraction and hybridization of Affymetrix microarrays. RNA was extracted from unfractionated liver samples and polysome fraction of samples separated on sucrose density gradient. To minimize biological variations, we pooled RNA from two perfused liver samples to use in each array analysis. The conditions were total and polysome fraction of Gcn2+/+, +Met or -Met; total and polysome fraction of Gcn2-/-, +Met or -Met. Each array analysis was done in duplicate.在真核生物中，对信使RNA（mRNA）翻译过程的调控，可实现基因产物快速、精准且精细调控的表达。在翻译流程中，翻译起始的首个阶段受到真核起始因子（eIFs）及其结合蛋白的严格调控。这11种真核起始因子可催化转运RNA（tRNA）、信使RNA（mRNA）与核糖体RNA（rRNA）结合形成功能性翻译复合物。其活性受到多种胞外与胞内信号的调控，信号类型涵盖全局调控信号（如激素信号、未折叠蛋白反应）以及特异性信号（如单氨基酸失衡、铁缺乏）。它们的调控作用兼具广泛性与特异性：广泛性体现为可上调或下调绝大多数细胞信使RNA的招募与翻译过程；特异性则体现为靶向调控带有特定调控特征的信使RNA翻译，例如5'端寡嘧啶序列（TOP）、上游开放阅读框（uORFs）或内部核糖体进入位点（IRES）。 在哺乳动物体内，有两条主要通路参与靶向性信使RNA翻译调控。雷帕霉素靶蛋白（TOR）激酶可诱导携带5'端寡嘧啶序列（TOP）的信使RNA及其他部分信使RNA子集的翻译；而eIF2激酶家族则对带有上游开放阅读框（uORFs）或内部核糖体进入位点（IRES）的信使RNA发挥类似调控作用。雷帕霉素靶蛋白（TOR）可靶向调控编码翻译相关蛋白的信使RNA的翻译，这一功能与其公认的调控细胞生长的作用一致。eIF2激酶家族的4个成员均可上调编码应激反应蛋白（如转录因子、分子伴侣）的信使RNA的翻译。尽管这4种激酶的主要底物均为eIF2，但已有研究文献显示，每种激酶在响应特定激活应激时，既存在共同的调控效应，也具有独特的作用特点。这表明激活的eIF2激酶可同时调控全局范围与特定子集的信使RNA翻译。 截至目前，鲜有研究验证这一假说，且尚未有针对哺乳动物的相关研究。本研究通过芯片分析技术，旨在检测应激反应后全局信使RNA向多聚核糖体（polysomes）的迁移情况，并对比两种eIF2α激酶——GCN2与PERK分别激活后的翻译响应差异。 实验总体设计：将Gcn2野生型或基因敲除小鼠的肝脏用完全氨基酸培养基或缺乏甲硫氨酸的培养基进行灌流，随后用于RNA提取及Affymetrix芯片的杂交检测。从未经分级分离的肝脏样本以及经蔗糖密度梯度离心分离得到的多聚核糖体组分中提取RNA。为最小化生物学差异，本研究将两份灌流肝脏样本的RNA混合后用于单次芯片分析。实验分组涵盖以下四类样本：Gcn2野生型（Gcn2+/+）小鼠的总RNA及多聚核糖体组分，分别经甲硫氨酸充足（+Met）或甲硫氨酸缺乏（-Met）处理；Gcn2基因敲除型（Gcn2-/-）小鼠的总RNA及多聚核糖体组分，同样分别经甲硫氨酸充足或缺乏处理。所有芯片分析均重复进行两次。