Uncovering the Role of UFM1 in the Release of Arrested Peptides from Stalled Ribosomes at the Endoplasmic Reticulum (ER) Membrane

摘要 核糖体相关质量控制(Rqc)是细胞处理长时间翻译停滞引起的过程。 由于遗传错误、带电tRNA不足或错误的mRNA导致核糖体碰撞和潜在的 细胞毒性，不完全，翻译产品。核糖体分裂成游离的40s和60s亚基，不完全 新生链(阻滞物或AP)作为一种多肽tRNA加合物，它阻碍60年代的出口隧道和P-位点。在……里面 胞浆是一个复杂的过程，细胞如何去除AP，回收60S亚单位，并降解 美联社然而，对内质网产生的蛋白质如何管理这一过程的相应研究 网状(ER)膜缺乏。胞质和ER-RQC之间的关键区别在于，在ER膜上， AP被整合到Sec61转运子中，从而与胞质RQC和降解机制分离。多么 细胞管理这种拓扑约束是这一提议的重点。Kopito实验室和其他实验室已经表明，ER 局部核糖体碰撞导致小泛素样蛋白UFM1与核糖体蛋白L26结合 (RPL26)在60S亚基上(UFM化)。UFM1基因敲除细胞(UFM1KO)阻止内质网蛋白酶体降解 定位的AP，但不是胞质AP，提示在ER-RQC中UFM化和AP降解之间存在联系。我假设 RPL26的UFM化可作为向停滞的ER核糖体募集蛋白质的标志，这是分解1960-AP-AP所必需的。 转化子复合体，从而使AP可以被降解。在本提案的目标1和目标2中，我将确定 使用两种互补方法进行UFM化：通过将生物素连接酶融合到UFM1进行邻近标记和亲和力 UFM1结合蛋白的纯化。这些实验是这样设计的，我将识别与之相互作用的蛋白质 结合的UFM1而不是游离的UFM1。初步结果表明，内质网定位的转位相关蛋白是 丰富的邻近标记实验支持UFM化发生在ER膜附近的模型 易位子。初步亲和纯化实验表明，已知的胞内RQC蛋白在 UFM化核糖体支持UFM化在ER-RQC中的作用。在本提案的目标3中，我将定义 UFM化导致AP降解的机制。我将首先评估确定的UFM化读者的角色 通过依美汀追逐和代谢脉冲/追赶实验，在目标1和目标2中稳定AP。我还将测试UFM化是否 促进AP从核糖体或转位蛋白释放。UFM化途径在大多数情况下是保守的 真核物种，对后生动物细胞的造血和神经发育是必不可少的。这个基因的突变 这一途径与多种人类疾病有关。尽管它很重要，但我们并不完全了解 这条路正在进行中。在这份提案中，我将揭示UFM化的读者，并更好地定义它在ER-RQC中的作用。一个 要了解UFM化缺陷是如何导致人类疾病的，更好的理解是必要的。

Abstract Ribosome-associated quality control (RQC) is the process by which cells deal with prolonged translational stalls caused by genetic errors, insufficient charged tRNA, or faulty mRNA which results in ribosome collisions and potentially cytotoxic, incomplete, translation products. Ribosomes are split into a free 40S and 60S subunit with an incomplete nascent chain (arrest product or AP) as a peptidyl tRNA adduct which obstructs the exit tunnel and P-site of the 60S. In the cytosol, an intricate process has been defined for how cells remove the AP, recycle the 60S subunit, and degrade the AP. However, corresponding research into how this process is managed for proteins produced at the endoplasmic reticulum (ER) membrane is lacking. The key difference between cytosolic and ER-RQC is that at the ER membrane the AP is integrated into the SEC61 translocon and thus separated from the cytosolic RQC and degradative machinery. How cells manage this topological restraint is the focus of this proposal. The Kopito lab and others have shown that ER localized ribosome collisions result in the conjugation of the small ubiquitin-like protein UFM1 to ribosomal protein L26 (RPL26) on the 60S subunit (UFMylation). UFM1 knockout cells (UFM1KO) prevent proteasomal degradation of ER localized, but not cytosolic, APs suggesting a link between UFMylation and AP degradation in ER-RQC. I hypothesize that UFMylation of RPL26 severs as mark to recruit proteins to stalled ER ribosomes necessary for resolving the 60S-AP- translcoon complex so that the AP may be degraded. In Aim 1 and Aim 2 of this proposal I will identify the readers of UFMylation using two complimentary approaches: proximity labeling via fusion of a biotin ligase to UFM1 and affinity purification of UFM1 binding proteins. These experiments are designed such that I will identify proteins that interact with conjugated UFM1 and not free UFM1. Preliminary results show that ER localized, translocon associated proteins, are enriched in proximity labeling experiments which supports the model that UFMylation occurs at the ER membrane near the translocon. Preliminary affinity purification experiments reveal that known cytosolic RQC proteins are enriched on UFMylated ribosomes supporting the role of UFMylation in ER-RQC. In Aim 3 of this proposal, I will define the mechanism by which UFMylation leads to AP degradation. I will first evaluate the role of UFMylation readers identified in Aims 1 and 2 in AP stability via emetine chase and metabolic pulse/chase experiments. I will also test if UFMylation facilitates release of the AP from the ribosome or translocon. The UFMylation pathway is conserved amongst most eukaryotic species and is essential for hematopoietic and neurological development in metazoan cells. Mutations in this pathway have been linked to a wide variety of human diseases. Despite its importance, we do not fully understand what this pathway is doing. In this proposal, I will uncover the readers of UFMylation and better define its role in ER-RQC. A better understanding is necessary to understand how defects in UFMylation lead to human disease.