The mechanism of Ire1-mediated mRNA cleavage during endoplasmic reticulum stress

 描述（由申请人提供）：未折叠蛋白反应（UPR）是一种重要的细胞信号传导系统，可检测内质网（ER）内错误折叠蛋白的积聚，并启动细胞反应以维持蛋白质稳态。UPR信号传导的异常可导致从2型糖尿病到神经系统疾病的多种疾病状态。UPR信号通路最古老的分支是通过ER定位的核糖核酸内切酶（RNase）Ire 1在ER应激时的激活而启动的。一旦被激活，Ire 1特异性地切割XBP 1的胞质mRNA，产生一种活性转录因子，驱动UPR靶基因的表达，以减轻ER应激。此外，本发明还提供了一种方法， Ire 1还通过受调节的Ire 1依赖性衰变（RIDD）途径混杂地切割ER定位的mRNA，以减少进入的蛋白质负荷的负担。虽然最近在了解Ire 1/UPR通路的分子基础方面取得了进展，但该通路的基本机制特征仍然知之甚少。本申请的目的是了解Ire 1如何选择性地发现和切割其mRNA底物，同时避免非特异性切割其他细胞mRNA。这些研究将引导我们发现UPR信号传导的新步骤，这些步骤可以被药物靶向治疗疾病。目前的研究建议分为两个具体目标：1。确定Ire 1的RNA酶活性在细胞中是如何调节的。我们最近发现Ire 1与ER膜中的Sec 61 translocon-核糖体形成复合物，促进其正确mRNA底物的切割。我们将首先测试的假设，这种复合物也可以防止非特异性RNase活性的Ire 1访问和裂解细胞溶质的mRNA通过使用RNA测序，生物化学和结构的方法。第二，我们将建立一个重组的蛋白脂质体系统，以测试Sec 61易位在调节Ire 1寡聚化，磷酸化和RNA酶活性在细胞中的作用。2.确定mRNA底物如何靶向ER膜中的Ire 1。我们已经表明，编码可溶性蛋白的XBP 1 mRNA利用SRP途径靶向Ire 1-Sec 61转位子复合物，以被Ire 1切割。这些研究和其他研究提出了两个重要问题。不遵循SRP途径的Ire 1底物mRNA如何靶向ER膜中的Ire 1？Ire 1 mRNA底物如何选择性靶向ER膜中的Ire 1-Sec 61转位子复合物，因为Ire 1相对于Sec 61是低丰度蛋白质？我们将结合核糖体分析和生化重建来解决这些问题。

 DESCRIPTION (provided by applicant): The unfolded protein response (UPR) is an essential cell signaling system that detects the accumulation of misfolded proteins within the endoplasmic reticulum (ER) and initiates a cellular response in order to maintain protein homeostasis. Aberrations in UPR signaling can lead to multitude of diseased states ranging from type 2 diabetes to neurological disorders. The most ancient branch of the UPR signaling pathways is initiated by activation of the ER-localized endoribonuclease (RNase) Ire1 upon ER stress. Once activated, Ire1 specifically cleaves the cytosolic mRNA of XBP1 to produce an active transcription factor that drives expression of UPR target genes to mitigate ER stress. In addition, Ire1 also promiscuously cleaves ER-localized mRNAs through the regulated Ire1-dependent decay (RIDD) pathway to reduce the burden of the incoming protein load. Although recent progress in understanding molecular basis of the Ire1/UPR pathway, fundamental mechanistic features of this pathway remain poorly understood. The goal of this application is to understand how Ire1 selectively finds and cleaves its mRNA substrates while avoiding non-specific cleavage of other cellular mRNAs. These studies will lead us to discover new steps in the UPR signaling, which can be targeted by drugs to treat diseases. The current research proposal is divided into two specific aims: 1. Determine how the RNase activity of Ire1 is regulated in cells. We have recently discovered that Ire1 is in a complex with the Sec61 translocon-ribosome in the ER membrane that facilitates cleavage of its correct mRNA substrates. We will first test the hypothesis that this complex may also prevent the non-specific RNase activity of Ire1 from accessing and cleaving cytosolic mRNAs by the use of RNA sequencing, biochemical and structural approaches. Second, we will establish a reconstituted proteoliposome system to test the role of the Sec61 translocon in regulating Ire1 oligomerization, phosphorylation and RNase activity in cells. 2. Determine how mRNA substrates are targeted to Ire1 in the ER membrane. We have shown that XBP1 mRNA, which encodes a soluble protein, utilizes the SRP pathway for targeting to the Ire1-Sec61 translocon complex for cleavage by Ire1. These and other studies raise two important questions. How are Ire1 substrate mRNAs that do not follow the SRP pathway targeted to Ire1 in the ER membrane? How Ire1 mRNA substrates are selectively targeted to Ire1-Sec61 translocon complexes in the ER membrane since Ire1 is a low abundant protein relative to Sec61? We will address these questions by a combination of ribosome profiling and biochemical reconstitution.