Identifying the components and properties of the new EIF5B RNP granule

我们最近发现了一个以前未知的过程，它增强了特定的翻译 通过形成一种生物凝集物而形成的mRNAs。到目前为止发现的许多颗粒都包含一个 核酸和蛋白质的混合物。在细胞质中，凝集物通常是核糖核蛋白(Rnp)。 颗粒。其中两个研究最好的是应力颗粒(SG)和加工体(P-Body)隔离 RNA，并与蛋白质合成的抑制有关。与先前识别出的这些相反 胞质颗粒这种新的生物分子凝聚物(5B颗粒)与刺激 翻译特定的mRNA。压力诱导剂，如亚砷酸钠治疗或山梨醇，刺激 5B颗粒的形成。有趣的是，这些相同的处理刺激了SG的形成并增加了 P小体的数量，表明在应激条件下，细胞既含有抑制性又含有刺激性 颗粒。在这些条件下，与应激颗粒相反，5B颗粒刺激IRES活性和 代表了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，我们提出了两个具体目标。 目的1.确定5B颗粒的成分并确定与其他成分的关系 胞质颗粒。我们将使用无偏见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学来鉴定与 EIF5B在有利于5B颗粒形成的条件下。我们将使用邻近标记和降水来富集 EIF5B相关蛋白。我们还将采用候选方法，使用其他颗粒的标记和 细胞质RNA结合蛋白测定应激颗粒、P小体和5B的关系 颗粒剂。 目的2.确定5B颗粒对翻译的整体影响我们将使用mRNA-seq， 多聚体图谱和核糖体保护片段(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 并分析翻译的全球影响。

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.