Identifying the components and properties of the new EIF5B RNP granule

我们最近发现了一种以前未知的过程，可以增强特定内容的翻译 mRNA通过形成生物冷凝物。迄今为止鉴定的许多颗粒含有 核酸和蛋白质的混合物。在细胞质中，凝聚物通常是核糖核蛋白（RNP） 颗粒。其中两个研究得最好的是应激颗粒（SG）和加工体（P-body）螯合剂 RNA和与蛋白质合成的抑制有关。与先前确定的 细胞质颗粒这种新的生物分子凝聚物（5 B颗粒）与刺激有关， 翻译特定的mRNA。应激诱导剂，如亚砷酸钠处理或山梨醇，刺激 形成5 B颗粒。有趣的是，这些相同的处理刺激SG形成并增加了 大量的P-体，表明在应激条件下，细胞既含有抑制性的，也含有刺激性的 颗粒。在这些条件下，与应激颗粒相反，5 B颗粒刺激IRES活性， 代表了一种新的细胞质凝聚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.确定5 B颗粒的组分并确定与其他组分的关系 胞质颗粒我们将使用无偏的方法和候选方法来鉴定蛋白质 5 B颗粒的成分。我们将使用定量质谱法来鉴定蛋白质， EIF 5 B在有利于5 B颗粒形成的条件下。我们将使用邻近标记和沉淀来富集 EIF 5 B相关蛋白。我们还将采用候选方法，使用其他颗粒的标记， 细胞质RNA结合蛋白，以确定压力颗粒，P-体和5 B的关系 颗粒。 目标2.确定5 B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 5 B颗粒的作用。我们为EIF 5 B创建了功能获得和功能丧失模型。此外，本发明还提供了一种方法， 我们已经确定了eIF 5 B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。

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.