Mapping new dimensions in gene expression regulation

程序摘要 错误折叠的蛋白质在内质网(ER)中积累，随后的氧化还原失衡激活了 未折叠蛋白质反应(UPR)，以恢复健康的细胞蛋白质组。普遍定期审议的失调是人类的关键 疾病，包括神经退行性变和癌症。UPR涉及基因表达调控的所有组成部分，即 转录、翻译，以及RNA和蛋白质的降解，这些都是高度协调的，遵循复杂的动态。在… 首先，翻译起始因子eif2α的磷酸化在激活的同时抑制了一般蛋白质的合成。 翻译编码ATF4等转录因子的特定mRNAs。磷酸化也激活了IRE1 剪接并激活XBP1转录因子的mRNA的核酸内切酶。XBP1和ATF4，以及ATF6 和其他转录因子，通过转录UPR和凋亡基因来触发第二波反应。此外， XBP1的活性不仅受IRE1控制，还受SUMO化和乙酰化的影响。IRE1反过来也可以降解 SUMO mRNA，下调这种翻译后修饰，并创建一种反馈调节机制。 这些过程伴随着广泛的RNA和蛋白质降解，去除了不可修复的分子，产生了 一个高度互联的系统。我们最近的工作使用了时间序列转录组学和蛋白质组学数据以及 统计模型显示，在内质网应激过程中，mRNAs以脉冲式方式变化，而蛋白质浓度 调整不那么迅速，看起来像是切换到新的稳定状态。 考虑到这些错综复杂的关系，我认为引入系统层面的分析来研究 并以全面和公正的方式评估参与其基因表达调控的过程。我的 实验室在定量质谱学、下一代测序、计算分析和 评估新机构的实验为这类工作提供了理想的背景。在未来五年，我们将 将单个时间点的分析转移到应激反应的轨迹；我们将从 浓度变化到估计RNA和蛋白质合成和降解的速度；我们将绘制蛋白质图谱 不同的翻译后修饰的位置和变化对这些比率的变化。这些努力将提供 向科学界提供宝贵的资源和统计工具。他们还将介绍监管原则。 在普遍定期审议期间，并支持具体的新的调查途径。我们才刚刚开始理解 差异化使用和修改翻译机械的部件，在未来五年内，我们将遵循 关于核糖体修饰、核糖体亚基的差异稳定性和选择性剪接的最新发现 应激条件下氨基酰-tRNA合成酶的活性。此外，即使是研究充分的因素，如ATF4，仍然是不完全的 我们将测试ATF4中的新序列元素和其他基因在其 对翻译的影响。最后，基因对变异的稳健性，例如突变，是理解细胞的核心 病理学，再一次，我们最近的工作表明，一些基因在某些频率上对变异很强，但在另一些频率上则不是。我们 将在有针对性的研究中检验这些假设。

Program Abstract Accumulation of misfolded proteins in the endoplasmic reticulum (ER) and subsequent redox imbalance activate the Unfolded Protein Response (UPR) to restore a healthy cellular proteome. Dysregulation of the UPR is key to human diseases, including neurodegeneration and cancer. The UPR involves all components of gene expression control, i.e. transcription, translation, and RNA and protein degradation, that are highly coordinated and follow intricate dynamics. At first, phosphorylation of the translation initiation factor eIF2α suppresses general protein synthesis while activating translation of specific mRNAs that encode transcription factors such as ATF4. Phosphorylation also activates the IRE1 endonuclease that splices and activates the mRNA of the XBP1 transcription factor. XBP1 and ATF4, together with ATF6 and other transcription factors, trigger a second wave of response by transcribing both UPR and apoptosis genes. Further, XBP1 activity is not only controlled by IRE1, but also by SUMOylation and acetylation. IRE1 in turn can also degrade SUMO mRNA, down-regulating this post-translational modification and creating a feedback mechanism of regulation. These processes are accompanied by extensive RNA and protein degradation that remove irreparable molecules, creating a highly interconnected system. Our recent work using time-series transcriptomics and proteomics data as well as statistical modeling showed that during ER stress, mRNAs change in a pulse-like manner, while protein concentrations adjust less rapidly and appear to switch to a new steady state. Given these intricate relationships, I argue that the time is ripe to introduce systems level analyses to studies of the UPR and assess the processes involved in its gene expression regulation in a comprehensive and unbiased way. My laboratory's expertise in quantitative mass spectrometry, next-generation sequencing, computational analysis, and experiments to evaluate new mechanisms provides the ideal background for such work. In the next five years, we will move analysis of single time points to trajectories of the stress response; we will progress from examination of concentration changes to estimating rates of RNA and protein synthesis and degradation; and we will map the protein position and changes of diverse post-translational modifications to changes in these rates. These efforts will provide valuable resources and statistics tools to the scientific community. They will also inform on regulatory principles during the UPR and support specific new routes of investigation. We are only beginning to understand the differential use and modification of components of the translation machinery, and in the next five years, we will follow up on our recent findings on ribosome modifications, differential stability of ribosome subunits, and alternative splicing of aminoacyl-tRNA synthetases under stress. Further, even well-studied factors such as ATF4 still are incompletely understood in their regulation, and we will test the role of new sequence elements in ATF4 and other genes in their effect on translation. Finally, the robustness of genes to variation, e.g. mutation, is central to understanding cellular pathology, and again, our recent work suggests that some genes are robust to variation in some rates, but not others. We will test these hypotheses in targeted studies.