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，可以增强特定的翻译 通过形成生物冷凝物的mRNA。迄今为止确定的许多颗粒包含一个 核酸和蛋白质的混合物。在细胞质中，冷凝水通常是核糖核蛋白（RNP） 颗粒。其中两个研究中的两个，应力颗粒（SG）和加工体（P-y）隔离器 RNA，与蛋白质合成的抑制有关。与这些先前确定的 细胞质颗粒这种新的生物分子冷凝物（5B颗粒）与刺激有关 特定mRNA的翻译。诱导诱导剂，例如砷酸钠或山梨糖醇，刺激 5B颗粒的形成。有趣的是，这些相同的治疗刺激了SG的形成，并增加了 P体数量的数量表明，在应力条件下，细胞含有抑制和刺激性 颗粒。在这些条件下，与应力颗粒相反，5B颗粒刺激IRES活性和 代表一种新的细胞质冷凝物。为了理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组成部分，以及这些颗粒有多大的影响全球翻译。 为此，我们提出了两个具体目标。 目标1。确定5B颗粒的组成部分，并确定与其他的关系 细胞质颗粒。我们将使用公正的方法和一种候选方法来识别蛋白质 5B颗粒的成分。我们将使用定量质谱法来鉴定与 在有利于5B颗粒形成的条件下EIF5B。我们将使用接近标签和降水来丰富 EIF5B相关蛋白。我们还将使用其他颗粒的标记来采用候选方法 细胞质RNA结合蛋白，以确定应力颗粒，P体和5B的关系 颗粒。 目标2。确定5b颗粒对翻译的全局效应，我们将使用mRNA-seq， 多元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创建了功能和功能模型的丧失。此外， 我们已经确定了促进颗粒形成的EIF5B的区域，使我们能够控制颗粒形成 并测定全球对翻译的影响。