Next generation gene expression analysis

摘要 错误折叠蛋白在内质网（ER）中的积累激活了未折叠蛋白反应（UPR） 其目的是恢复健康的细胞蛋白质组。UPR的失调是疾病的关键，例如神经变性。 为了发挥这一核心作用，普遍定期审议形成了一个过程网络，涉及复杂的转录、翻译和RNA， 蛋白质降解变化。例如，虽然普遍定期审议关闭了全球翻译，但它激活了特定的反应， 基因，如转录因子ATF 4。我们的目标是研究这个反应网络。 我们从几个角度研究了这个网络：分析哺乳动物UPR的动态，我们发现 数百个基因的调控信号我们在ATF 4中发现了一个翻译调控元件， 在胁迫条件下基因的翻译诱导被忽略了。该元件由起始和终止密码子组成， 阻止核糖体。我们在数百个富含信号分子的基因中发现了起始点。此外，我们还侧写了 蛋白质修饰，例如泛素化，以响应压力。此外，我们开始比较两个国家的普遍定期审议情况， 相关的运动神经元具有不同的应力敏感性，这与它们在肌萎缩侧索硬化症中的作用一致 (ALS)：在ALS期间，应激敏感的脊髓运动神经元死亡较早，而更多的应激抵抗的颅运动神经元 直到疾病的晚期。我们确定了分子特征，例如在蛋白酶体中，可以解释 运动神经元的差异应力敏感性。这样做，我们创建了用于综合分析的工具和资源。 在接下来的五年里，我们将解决这些发现中出现的三个主要问题：i）细胞如何在 一般来说，诱导应激反应基因，而一般翻译停止？（二）蛋白质修饰的作用 普遍定期审议？以及iii）翻译、蛋白质修饰和其他途径如何形成有效和稳健的反应 在压力下恢复蛋白质组健康的网络？具体来说，我们将研究启动-停止和其他 DROSHA和RAD 23 B的翻译调控元件，其在miRNA途径和DNA损伤中起作用 修复，分别，而且还链接到ER应激反应。使用功能增益构造，我们将对 启动-停止功能机制，并鉴定影响转录本定位、稳定性 和下游重新启动。我们将通过大规模的核糖体扫描评估来补充这些分析， 起始、转录稳定性的变化以及响应于应激而与mRNA结合的蛋白质的变化。第二条研究途径 将研究蛋白质ufmylation，一种与UPR和ER维持相关的泛素样蛋白质修饰， 翻译和DNA损伤反应。我们以前发现Ufl 1，一个关键的ufmylation基因，表达不同的 在颅和脊髓运动神经元的亚型，我们提出的工作将调查差异Ufl 1的影响， UPR在两个运动神经元中的表达。作为对这些分析的补充，我们将尝试识别新的和 神经元类型特异性ufmylation靶点，并研究它们与翻译调节，核糖体质量控制， DNA损伤修复这项工作将利用我们在系统规模和目标分析方面的专业知识， Proteostasis网络的特性。

Abstract Accumulation of misfolded proteins in the endoplasmic reticulum (ER) activates the Unfolded Protein Response (UPR) which aims at restoring a healthy cellular proteome. Dysregulation of the UPR is key to diseases, e.g. neurodegeneration. For this central role, the UPR forms a network of processes, involving complex transcription, translation, and RNA and protein degradation changes. For example, while the UPR shuts down global translation, it activates specific response genes, such as the transcription factor ATF4. Our goal is to investigate this response network. We have investigated this network from several angles: profiling the dynamics of the mammalian UPR, we identified regulatory signatures for hundreds of genes. We discovered a translation regulatory element in ATF4 whose role in translation induction of the gene under stress had been overlooked. The element consists of a start and stop codon and stalls ribosomes. We discovered start-stops in hundreds of genes enriched for signaling molecules. In addition, we profiled protein modifications, e.g. ubiquitination, in response to stress. Further, we began to compare the UPR in two closely related motor neurons with differential stress-sensitivity that is consistent with their role in Amyotrophic Lateral Sclerosis (ALS): stress-sensitive spinal motor neurons die early during ALS, while more stress-resistant cranial motor neurons survive until late stages of the disease. We identified molecular signatures, e.g. in the proteasome, that can explain the motor neurons’ differential stress sensitivity. Doing so, we created tools and resources for integrative analysis. In the next five years, we will address three major questions that arise from these findings: i) How does the cell, in general, induce stress response genes while general translation is halted? ii) What are the roles of protein modifications in the UPR? and iii) How do translation, protein modifications, and other pathways form an efficient and robust response network that restores proteome health upon stress? Specifically, we will investigate the role of start-stops and other elements in translation regulation of DROSHA and RAD23B, which function in the miRNA pathway and DNA damage repair, respectively, but also link to the ER stress response. Using a gain-of-function construct, we will deconvolute the mechanism of start-stop function and identify regulators of ribosome stalling that affect transcript localization, stability, and downstream re-initiation. We will complement these analyses with large-scale assessment of ribosome scanning and initiation, changes in transcript stability and in proteins bound to mRNAs in response to stress. A second research avenue will investigate protein ufmylation, a ubiquitin-like protein modification linked to the UPR and ER maintenance, but also to translation and the DNA damage response. We previously found that Ufl1, a key ufmylation gene, expresses different isoforms in cranial and spinal motor neurons, and our proposed work will investigate the impact of differential Ufl1 expression on the UPR in the two motor neurons. Complementing these analyses, we will attempt to identify novel and neuron type specific ufmylation targets and investigate their links to translation regulation, ribosome quality control, and DNA damage repair. The work will exploit our expertise in systems-scale and targeted analysis to understand new properties of the proteostasis network.