Ribosome stalling and activation of stress responses

项目摘要/摘要 在蛋白质合成过程中，核糖体整合了多个线索，以确保在 正确的地点、正确的时间和正确的注意力。这些提示是由变化触发的信号的结果 细胞需求和环境条件，如增殖和应激。在真核生物中，整合应激 应答(Isr)通过激活作用于启动因子eif2的激酶来响应压力。 EIF2的磷酸化抑制了全球翻译，但也降低了关键的支持生存的翻译 MRNAs。在酵母中，isr被eif2激酶GCN2激活。最近来自几个组织的研究，包括 我们的研究指出了核糖体的核心作用，特别是它们在ISR激活期间的停滞。 有趣的是，核糖体停滞也激活了核糖体质量控制(RQC)，这严重依赖于E3 连接酶Hel2。在之前的资金支持期间，我们证实了核糖体激活了Hel2 碰撞，并表明，化学侮辱，破坏RNA触发RQC。值得注意的是，这些特工 还要激活ISR，这表明RQC和ISR是紧密协调的。在最近的一项研究中，我们展示了 核糖体碰撞不仅激活了这两个过程，而且其中一个过程的激活抑制了 其他的。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，在提交的初步数据中 在这项提案中，我们提供了令人信服的证据，证明核糖体碰撞在向其他核发出信号中起到了作用。 酸损伤途径，特别是那些参与DNA损伤修复的途径。这项提案的重点是 了解相互碰撞的核糖体可以激活这些看似无关的 流程。我们的初步数据表明，核糖体的A状态对于ISR激活是重要的，并且在 目的1我们将探索核糖体在不同应激条件下的构象并评估它们如何影响 GCN2招聘。我们将通过重组ISR和RQC活动来扩展这些研究，以提供 RQC明显优先于ISR激活的机理理解。值得注意的是，健壮的ISR还 需要高度保守的转录辅助激活因子Mbf1的存在，我们和其他人向 结合碰撞的核糖体。在目标2中，我们将使用双管齐下的方法检验这一假设，即拖延会激活ISR 机制，其中碰撞的核糖体除了激活GCN2外，还调节了Mbf1对ISR的共激活。在……里面 特别是，我们将剖析Mbf1与核糖体的相互作用在调节其功能中的作用。 翻译修饰。最后，我们有大量的初步数据将RNA质量控制过程联系起来 用DNA修复。目标3确定两者之间信号转导的分子细节 过程，到目前为止还没有被探索过。总之，我们将利用我们在核糖体生物化学和 酵母遗传学与我们在过去资助期积累的资源相结合，以揭示如何碰撞 核糖体为几个保守的信号过程提供了一个结构平台。

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.