Ribosome stalling and activation of stress responses

项目总结/摘要 在蛋白质合成过程中，核糖体整合了多种信号，以确保正确的蛋白质是在蛋白质合成过程中产生的。 在正确的时间，正确的地点，正确的浓度这些线索是由不同的信号触发的结果 细胞需要和环境条件，如增殖和压力。在真核生物中， 应激反应（ISR）通过激活作用于起始因子eIF 2 β的激酶来应答应激。 eIF 2 β的磷酸化抑制了整体翻译，但也解除了关键促生存因子的翻译。 mRNA。在酵母中，ISR被eIF 2 β激酶Gcn 2激活。几个团体最近的研究，包括 我们的研究已经指出了核糖体的核心作用，特别是它们在ISR激活过程中的停滞。 有趣的是，核糖体停滞也激活核糖体质量控制（RQC），这主要取决于E3 连接酶Hel 2。在上一个资助期间，我们确定了Hel 2是响应于核糖体激活的。 碰撞，并表明化学损伤，破坏RNA触发RQC。值得注意的是，这些特工 并激活ISR，表明RQC和ISR紧密协调。在最近的一项研究中， 不仅核糖体碰撞激活了这两个过程，而且其中一个过程的激活抑制了另一个过程的激活。 其他.从这些研究中发现，碰撞的核糖体被广泛用作传感器， 根据压力的类型和程度，触发适当的反应。事实上，在初步数据中， 在这个提议中，我们提供了令人信服证据，证明核糖体碰撞在向其他核 酸损伤途径，特别是参与DNA损伤修复的途径。该提案的重点是 了解碰撞的核糖体可以激活这些看似无关的分子原理 流程.我们的初步数据表明，核糖体的A状态对ISR激活很重要， 目的1我们将探索核糖体的构象在不同的压力条件下，并评估它们如何影响 GCN 2招聘我们将通过重组ISR和RQC活动来扩展这些研究， 对RQC比ISR明显优先活化的机理理解。值得注意的是，强大的ISR还 需要高度保守的转录辅激活因子Mbf 1的存在，我们和其他人表明， 结合碰撞的核糖体。在目标2中，我们将使用一个双管齐下的方法来测试失速激活ISR的假设。 机制，其中碰撞的核糖体除了激活Gcn 2调节ISR共激活Mbf 1。在 特别是，我们将剖析Mbf 1与核糖体相互作用的作用，通过后 翻译修饰最后，我们有丰富的初步数据连接RNA质量控制过程 DNA修复。目的3建立关于两者之间信号转导的分子细节 这是迄今为止尚未探索的过程。总之，我们将利用我们在核糖体生物化学方面的专业知识， 酵母遗传学结合我们在过去的资助期间积累的资源，以揭示如何碰撞 核糖体为几种保守的信号传导过程提供了结构平台。

PROJECT SUMMARY/ABSTRACT During protein synthesis, the ribosome integrates multiple cues to ensure that the correct protein is made at the right place, the right time and at the right concentration. These cues are the result of signals triggered by varying cellular needs and environmental conditions such as proliferation and stress. In eukaryotes, the integrated-stress response (ISR) responds to stresses through the activation of kinases that act on the initiation factor eIF2. Phosphorylation of eIF2 represses global translation, but also derepresses translation of key pro-survival mRNAs. In yeast, ISR is activated by the eIF2 kinase Gcn2. Recent studies from several groups, including ours, have pointed to a central role for ribosomes and in particular their stalling during the activation of ISR. Interestingly, ribosome stalling also activates ribosome-quality control (RQC), which depends critically on an E3 ligase Hel2. During the previous funding period, we established that Hel2 is activated in response to ribosome collisions and showed that chemical insults that damage RNA trigger RQC. Notably, these very same agents also activate ISR, suggesting that RQC and ISR are tightly be coordinated. In a very recent study, we showed that not only do ribosome collisions activate both processes, but that the activation of one suppresses that of the other. Emerging from these studies is the observation that collided ribosomes are widely used as sensors to trigger an appropriate response, depending on the type and level of stress. Indeed, in preliminary data presented in this proposal, we provide compelling evidence for a role for ribosome collisions in signaling to other nucleic acid damage pathways, particular those involved in DNA-damage repair. This proposal is focused on understanding the molecular rationale by which collided ribosomes can activate these seemingly unrelated processes. Our preliminary data indicate that the A status of the ribosome is important for ISR activation, and in Aim 1 we will probe the conformation of ribosomes under various stress conditions and assess how they impact Gcn2 recruitment. We will expand on these studies by reconstituting ISR and RQC activities to provide a mechanistic understanding for the apparent preferential activation of RQC over ISR. Notably, robust ISR also requires the presence of the highly conserved transcriptional coactivator Mbf1, which we and others showed to bind collided ribosomes. In Aim 2, we will test the hypothesis that stalling activates ISR using a two-pronged mechanism, in which collided ribosomes in addition to activating Gcn2 modulate ISR coactivation by Mbf1. In particular, we will dissect the role of Mbf1 interactions with the ribosome in regulating its function through post- translational modification. Finally, we have a wealth of preliminary data linking RNA-quality control processes with DNA repair. Aim 3 establishing molecular details about how signaling is transduced between the two processes, which is hitherto unexplored. Altogether, we will leverage our expertise in ribosome biochemistry and yeast genetics in combination with resources we accrued over the past funding period to reveal how collided ribosomes provide a structural platform for several conserved signaling processes.