Mechanism and Regulation of eIF6 in Translation

eIF6翻译机制及调控

• 批准号: 10444189
• 负责人: Sofia Origanti
• 金额: $ 31.12万
• 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/10444189
• 关键词: Address; Affect; Allosteric Regulation; Aminoacyl-tRNA hydrolase; Binding; Biochemical; Biological Assay; Biological Models; Biophysics; Cell Cycle; Cell Nucleolus; Cell Survival; Cells; Cellular Assay; Childhood Leukemia; Colon Carcinoma; Complex; Data; Deuterium; Disease; Disease Progression; Dissociation; Ensure; Eukaryotic Initiation Factors; Event; GTP Binding; Generations; Goals; Growth; Growth and Development function; Guanosine Triphosphate Phosphohydrolases; Human; Hydrogen; Hydrolysis; Impairment; Inherited; Link; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Messenger RNA; Metabolic Diseases; Methodology; Mitosis; Modeling; Molecular; Mutation; Pathogenesis; Pathologic; Patients; Peptide Initiation Factors; Phenotype; Phosphorylation; Physiological; Process; Prognosis; Proliferating; Protein Biosynthesis; Protein Deregulation; Quality Control; RPL10 gene; Regulation; Ribonucleoproteins; Ribosomal RNA; Ribosomes; Role; Shwachman-Diamond syndrome; Site; Tail; Testing; Therapeutic; Time; Tissue Microarray; Trans-Activators; Translation Initiation; Translations; Tumor-Derived; Up-Regulation; Work; aurora B kinase; base; cancer cell; cancer subtypes; cancer type; experimental study; in vivo; insight; leukemia; malignant breast neoplasm; mouse model; mutant; new therapeutic target; novel; overexpression; release factor; single molecule; therapeutic development; therapeutic target; triple-negative invasive breast carcinoma; tumorigenesis

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.

项目总结 核糖体是大的核糖核蛋白复合体，对翻译控制是不可或缺的。该组件和 核糖体亚基的成熟涉及许多反式作用因子，这些反式作用因子使亚基 翻译能力强。60S核糖体亚基的成熟是通过释放真核生物来完成的 启动因子-6(EIF6)和成熟因子-SBDS和EFL1-GTP酶的辅助作用。EIF6立体阻碍 60S和40S亚基的结合，因此，从60S释放它对于允许相互作用至关重要 在60s和mRNA结合的40s亚基之间。此外，eIF6在rRNA的加工中是必不可少的 核仁，并与翻译停滞的60S-核糖体质量控制复合体有关。鉴于其 EIF6活动的基本作用、空间和时间方面及其从60年代开始的释放必须密切相关 进行规范，以确保成功启动翻译。EIF6的释放受损是某些 核糖体疾病：Shwachman-Diamond综合征和RPL10突变驱动的儿童白血病。EIF6级别 在几种癌症中也是失控的，其表达增强与预后不良有关。 值得注意的是，限制eIF6水平可以抑制某些癌症的生长，而不会影响正常生长。因此， 靶向eIF6并从60年代开始释放eIF6被认为是治疗癌症的理想策略 以及核糖体疾病。然而，我们还不知道eif6在调节60s的不同步骤中所起的作用。 组装和成熟。此外，调节eIF6与60s相互作用的分子机制也不是 完全理解。为了理解这些机制，我们最近的工作确定了关键的残留物 在调节其与60S相互作用的eIF6的关键界面上，我们的初步数据提供了直接的 有证据表明，这种相互作用的破坏对癌细胞的生存是有害的。我们还确定了一部小说 在eIF6的C-末端的调控位点，并揭示了它对控制翻译速率的重要性。 在这些发现的基础上，在当前的提案中，我们的目标是定义促进 SBDS从不同的功能状态释放EIF6和EFl1以及揭示GTPase的作用 使用一套严格的生化、生物物理和单分子方法来研究EFl1的活性。此外, 我们的目标是揭示eIF6 C-末端对变构的调节机制，并阐明其功能 以及使用细胞和体内方法的疾病特异性eIF6突变的表型效应。这些研究 将提供对1960年代动力学的关键机械洞察力，并将使治疗学的发展成为可能 重点关注eIF6及其监管机构。