Mapping new dimensions in gene expression regulation

程序摘要 内质网（ER）中错误折叠蛋白质的积累和随后的氧化还原失衡激活了 未折叠蛋白质反应（UPR）恢复健康的细胞蛋白质组。普遍定期审议的失调是人类 疾病，包括神经变性和癌症。UPR涉及基因表达控制的所有组成部分，即 转录，翻译，RNA和蛋白质降解，这些都是高度协调的，并遵循复杂的动力学。在 首先，翻译起始因子eIF 2 α的磷酸化抑制一般蛋白质合成，同时激活 翻译特定的mRNA，编码转录因子，如ATF 4。磷酸化也激活IRE 1 在一些实施方案中，转录因子是剪接和激活XBP 1转录因子的mRNA的核酸内切酶。XBP 1和ATF 4以及ATF 6 和其他转录因子，通过转录UPR和凋亡基因触发第二波反应。此外，本发明还 XBP 1的活性不仅受IRE 1的调控，还受SUMO化和乙酰化的调控。IRE 1反过来也可以降解 SUMO mRNA，下调这种翻译后修饰并产生反馈调节机制。 这些过程伴随着广泛的RNA和蛋白质降解，去除不可修复的分子， 一个高度互联的系统。我们最近的工作使用时间序列转录组学和蛋白质组学数据， 统计模型显示，在内质网应激期间，mRNA以脉冲样方式变化，而蛋白质浓度 调整得不那么快，似乎切换到一个新的稳定状态。 鉴于这些错综复杂的关系，我认为，时机已经成熟，引入系统水平的分析，以研究 UPR，并以全面和公正的方式评估其基因表达调控所涉及的过程。我 实验室在定量质谱、下一代测序、计算分析和 评估新机制的实验为此类工作提供了理想的背景。未来五年，我们将 将单个时间点的分析移动到应激反应的轨迹;我们将从检查 浓度变化来估计RNA和蛋白质的合成和降解速率;我们将绘制蛋白质 位置和变化的各种翻译后修饰的变化，这些利率。这些努力将提供 科学界的宝贵资源和统计工具。他们还将通报监管原则 在普遍定期审议期间，支持具体的新调查路线。我们才刚刚开始了解 差异化使用和修改翻译机器的组件，在未来五年内，我们将遵循 我们最近在核糖体修饰、核糖体亚基的差异稳定性和可变剪接方面的发现 氨酰-tRNA合成酶在压力下的变化。此外，即使是充分研究的因素，如ATF 4仍然是不完全的， 我们将测试ATF 4和其他基因中新序列元件在其调控中的作用， 对翻译的影响。最后，基因对变异（例如突变）的鲁棒性是理解细胞遗传学的核心。 我们最近的研究再次表明，某些基因对某些比率的变化具有鲁棒性，但对另一些则不然。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.