Identifying the components and properties of the new EIF5B RNP granule

确定新型 EIF5B RNP 颗粒的成分和特性

• 批准号: 10646683
• 负责人: MICHAEL Thomas MARR
• 金额: $ 43.35万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-15 至 2025-09-14
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10646683
• 关键词: Address; Aging; Alzheimer' s Disease; Amyotrophic Lateral Sclerosis; Binding; Biological; Biological Assay; Cell Cycle; Cell physiology; Cells; Cellular Stress; Code; Cytoplasm; Cytoplasmic Granules; Disease; Elements; Gene Expression; Genome; Homeostasis; Initiator tRNA; Internal Ribosome Entry Site; Label; Liquid substance; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Messenger RNA; Modeling; Myopathy; Names; Nucleic Acids; Peptide Initiation Factors; Phase; Physical condensation; Precipitation; Process; Property; Protein Region; Protein Synthesis Inhibition; Proteins; RNA; RNA-Binding Proteins; Regulation; Repression; Ribonucleoproteins; Ribosomes; Role; Signal Transduction; Sorbitol; Specificity; Stress; System; Transcript; Translational Regulation; Translations; Viral; Work; eukaryotic initiation factor-5B; gain of function; human disease; interest; loss of function; mRNA capping; mRNA sequencing; polysome profiling; proteostasis; sodium arsenite; stress granule

We have recently uncovered a previously unknown process that enhances the translation of specific mRNAs through the formation of a biological condensate. Many of the granules identified to date contain a mixture of nucleic acids and proteins. In the cytoplasm, often the condensates are ribonucleoprotein (RNP) granules. Two of the best studied of these, the stress granule (SG) and the Processing body (P-body) sequester RNAs and are associated with the inhibition of protein synthesis. In contrast to these previously identified cytoplasmic granules this new biomolecular condensate (5B granule) is associated with the stimulation of translation of specific mRNAs. Stress inducing agents, such as sodium arsenite treatment or sorbitol, stimulate the formation of 5B granules. Interestingly, these same treatments stimulate SG formation and increase the number of P-bodies, suggesting that under stress conditions the cell contains both repressive and stimulatory granules. Under these conditions, and in contrast to stress granules, the 5B granules stimulate IRES activity and represent a new cytoplasmic condensate. In order to understand and start to explore what these granules are we need to know what components are in the granules and how much these granules influence global translation. To this end we are proposing two specific aims. Aim 1. Identify the components of the 5B granule and determine the relationship to other cytoplasmic granules. We will use an unbiased approach and a candidate approach to identify the protein components of the 5B granule. We will use quantitative mass spectrometry to identify proteins in proximity with EIF5B under conditions that favor 5B granule formation. We will use proximity labeling and precipitation to enrich EIF5B associated proteins. We will also take a candidate approach, using markers of the other granules and cytoplasmic RNA binding proteins to determine the relationship of the stress granule, the P-body and the 5B granule. Aim 2. Determine the global effect of the 5B granule on the translation We will use mRNA-seq, polysome profiling, and ribosome protected fragment (RPF) sequencing to determine the extent and specificity of the role of the 5B granule. We have created gain of function and loss of function models for EIF5B. In addition, we have identified the regions of eIF5B that promote granule formation allowing us to control granule formation and assay global effects on translation.

我们最近发现了一个以前未知的过程，可以增强特定内容的翻译 mRNAs通过形成生物凝结物。迄今为止发现的许多颗粒都含有 核酸和蛋白质的混合物。在细胞质中，缩合物通常是核糖核蛋白（RNP） 颗粒。其中研究最好的两个是应力颗粒 (SG) 和处理体 (P 体) 隔离器 RNA 和 与蛋白质合成的抑制有关。与之前确定的这些相比 细胞质颗粒这种新的生物分子凝聚物（5B颗粒）与刺激 特定 mRNA 的翻译。应激诱导剂，如亚砷酸钠处理或山梨糖醇，会刺激 5B颗粒的形成。有趣的是，这些相同的治疗方法可以刺激 SG 形成并增加 P 体的数量，表明在应激条件下，细胞同时包含抑制性和刺激性 颗粒。在这些条件下，与应激颗粒相反，5B 颗粒会刺激 IRES 活性并 代表新的细胞质凝聚物。为了了解并开始探索这些颗粒是什么 我们需要知道颗粒中有哪些成分以及这些颗粒对全局翻译的影响有多大。 为此，我们提出两个具体目标。 目标 1. 识别 5B 颗粒的成分并确定与其他颗粒的关系 细胞质颗粒。我们将使用公正的方法和候选方法来鉴定蛋白质 5B颗粒的成分。我们将使用定量质谱法来识别邻近的蛋白质 EIF5B 在有利于 5B 颗粒形成的条件下。我们将使用邻近标记和沉淀来富集 EIF5B 相关蛋白。我们还将采取一种候选方法，使用其他颗粒的标记和 细胞质 RNA 结合蛋白以确定应激颗粒、P 体和 5B 的关系 颗粒。 目标 2. 确定 5B 颗粒对翻译的整体影响 我们将使用 mRNA-seq， 多核糖体分析和核糖体保护片段 (RPF) 测序以确定程度和特异性 5B颗粒的作用。我们为 EIF5B 创建了功能增益和功能损失模型。此外， 我们已经确定了 eIF5B 促进颗粒形成的区域，使我们能够控制颗粒形成 并分析对翻译的全局影响。