Next generation gene expression analysis

摘要 错误折叠的蛋白在内质网(ER)中的积累激活了未折叠蛋白反应(UPR) 旨在恢复健康的细胞蛋白质组。UPR的失调是疾病的关键，例如神经退行性变。 对于这一核心作用，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以及 蛋白质降解会发生变化。例如，虽然普遍定期审议关闭了全球翻译，但它激活了特定的响应 基因，如转录因子ATF4。我们的目标是调查这个反应网络。 我们从几个角度研究了这个网络：分析哺乳动物UPR的动力学，我们发现 数百个基因的监管签名。我们在ATF4中发现了一个翻译调节元件，它在 该基因在应激状态下的翻译诱导被忽视。该元素由起始密码子和终止密码子组成 使核糖体停滞。我们在数百个富含信号分子的基因中发现了起止点。此外，我们还分析了 蛋白质修饰，例如泛素化，以响应压力。此外，我们开始将普遍定期审议分成两部分进行密切比较。 肌萎缩侧索硬化症中具有不同应激敏感性的相关运动神经元 (ALS)：应激敏感的脊髓运动神经元在ALS期间过早死亡，而更具抗应激能力的脑运动神经元 能活到疾病的晚期。我们确定了分子特征，例如在蛋白酶体中，可以解释 运动神经元的差值应激敏感性。为此，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产生的三个主要问题：i)细胞如何在 一般情况下，诱导应激反应基因，而一般翻译被停止？二)蛋白质修饰在以下过程中起什么作用 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强大的反应 在压力下恢复蛋白质组健康的网络？具体地说，我们将调查启停和其他 DROSHA和RAD23B在miRNA途径和DNA损伤中的翻译调控元件 修复分别还与内质网应激反应有关。使用函数增益构造，我们将去卷积 启动-停止功能机制和确定影响转录定位、稳定性和核糖体停滞调节因子， 以及下游的重新启动。我们将用核糖体扫描和大规模评估来补充这些分析 启动，转录稳定性的变化和与mRNAs结合的蛋白质对胁迫的反应。第二条研究途径 将研究蛋白质ufm化，一种泛素样蛋白修饰，与UPR和ER的维持有关，但也 翻译和DNA损伤反应。我们先前发现Ufl1，一个关键的ufm化基因，表达不同的 脑和脊髓运动神经元中的异构体，我们计划的工作将调查差异Ufl1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，我们将尝试确定新的和 神经元类型特异性超甲基化靶标，并研究它们与翻译调控、核糖体质量控制和 DNA损伤修复。这项工作将利用我们在系统规模和有针对性的分析方面的专业知识来理解新的 蛋白质平衡网络的特性。

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.