Mechanism and Regulation of eIF6 in Translation

eIF6翻译机制及调控

• 批准号: 10798681
• 负责人: Sofia Origanti
• 金额: $ 16.68万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-07 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10798681
• 关键词: Affect; Allosteric Regulation; Binding; Biochemical; Biophysics; Cell Nucleolus; Cell Survival; Childhood Leukemia; Complex; Data; Disease; Ensure; Eukaryotic Initiation Factors; Growth; Guanosine Triphosphate Phosphohydrolases; Impairment; Malignant Neoplasms; Messenger RNA; Metabolic Diseases; Molecular; Mutation; Phenotype; Prognosis; Protein Biosynthesis; Protein Deregulation; Quality Control; RPL10 gene; Regulation; Ribonucleoproteins; Ribosomal RNA; Ribosomes; Role; Shwachman-Diamond syndrome; Site; Therapeutic; Trans-Activators; Translation Initiation; Translation Process; Translations; Work; cancer cell; in vivo; insight; new therapeutic target; novel; ribosomopathy; single molecule; therapeutic development

PROJECT SUMMARY Ribosomes are large ribonucleoprotein complexes that are integral to translational control. The assembly and maturation of ribosomal subunits involves a multitude of trans-acting factors that render the subunits translationally competent. Maturation of the 60S ribosomal subunit is accomplished by the release of eukaryotic initiation factor-6 (eIF6) with the help of the maturation factors-SBDS and EFL1-GTPase. eIF6 sterically hinders association of the 60S and 40S subunits and therefore, its release from 60S is critical to permit interactions between 60S and the mRNA-bound 40S subunits. In addition, eIF6 is essential for rRNA processing in the nucleolus and is associated with the translationally stalled 60S-ribosome quality control complex. Given its essential roles, the spatial and temporal aspects of eIF6 activities and its release from 60S must be tightly regulated to ensure successful initiation of translation. Impaired release of eIF6 is the defining hallmark of certain ribosomopathies: Shwachman-Diamond syndrome and RPL10 mutations-driven pediatric leukemias. eIF6 levels are also deregulated in several cancers and its enhanced expression is associated with a poor prognosis. Remarkably, restricting eIF6 levels inhibits growth of certain cancers without affecting normal growth. Therefore, targeting eIF6 and its release from 60S has been proposed to be a desirable therapeutic strategy for cancers and ribosomopathies. However, we are yet to understand the role of eIF6 in modulating the distinct steps of 60S assembly and maturation. Also, the molecular mechanisms that regulate eIF6 interactions with the 60S are not completely understood. Towards understanding these mechanisms, our recent work has identified key residues in critical interfaces of eIF6 that modulate its interaction with 60S, and our preliminary data provide direct evidence that the disruption of this interaction is detrimental to cancer cell viability. We have also identified novel sites of regulation in the C-terminus of eIF6 and have uncovered its importance for controlling translational rates. Building on these discoveries, in the current proposal we aim to define the mechanistic steps that promote the release of eIF6 by SBDS, and EFL1 from distinct functional states of 60S and to uncover the role of GTPase activity of EFL1 using a rigorous set of biochemical, biophysical, and single molecule approaches. In addition, we aim to uncover the mechanism of allosteric regulation by the C-terminus of eIF6 and elucidate the functional and phenotypic effects of disease-specific mutations of eIF6 using cellular and in vivo approaches. These studies will provide crucial mechanistic insight into 60S dynamics and will enable the development of therapeutics focused on eIF6 and its regulators.

项目概要 核糖体是大型核糖核蛋白复合物，是翻译控制不可或缺的一部分。装配和 核糖体亚基的成熟涉及多种反式作用因子，使亚基 具有翻译能力。 60S核糖体亚基的成熟是通过释放真核细胞来完成的 起始因子 6 (eIF6) 在成熟因子 SBDS 和 EFL1-GTPase 的帮助下。 eIF6 空间位阻 60S 和 40S 亚基的关联，因此，它从 60S 的释放对于允许相互作用至关重要 60S 和 mRNA 结合的 40S 亚基之间。此外，eIF6 对于 rRNA 加工至关重要。 核仁并与翻译停滞的 60S-核糖体质量控制复合体相关。鉴于其 eIF6 活动的空间和时间方面及其从 60S 的释放必须紧密结合 进行监管以确保翻译的成功启动。 eIF6 释放受损是某些疾病的标志 核糖病：Shwachman-Diamond 综合征和 RPL10 突变驱动的儿童白血病。 eIF6 水平 在几种癌症中也失调，其表达增强与不良预后相关。 值得注意的是，限制 eIF6 水平可以抑制某些癌症的生长，而不影响正常生长。所以， 靶向 eIF6 及其从 60S 中的释放已被认为是一种理想的癌症治疗策略 和核糖体病。然而，我们尚未了解 eIF6 在调节 60S 的不同步骤中的作用 组装和成熟。此外，调节 eIF6 与 60S 相互作用的分子机制并不明确。 完全明白了。为了理解这些机制，我们最近的工作已经确定了关键残留物 在 eIF6 的关键接口中调节其与 60S 的相互作用，我们的初步数据提供了直接 有证据表明，这种相互作用的破坏不利于癌细胞的生存。我们还发现了新颖的 eIF6 C 末端的调控位点，并揭示了其对于控制翻译速率的重要性。 基于这些发现，在当前的提案中，我们的目标是定义促进 SBDS 和 EFL1 从 60S 的不同功能状态释放 eIF6，并揭示 GTPase 的作用 使用一套严格的生化、生物物理和单分子方法来检测 EFL1 的活性。此外， 我们的目标是揭示 eIF6 C 端变构调节的机制，并阐明其功能 使用细胞和体内方法研究 eIF6 疾病特异性突变的表型效应。这些研究 将为 60S 动力学提供重要的机制见解，并将促进治疗学的发展 重点关注 eIF6 及其监管机构。