Small molecule effectors of maternal gene expression in C. elegans embryogenesis

描述(由申请人提供)：提出了一种内聚性策略来识别母体基因表达的小分子效应器。在卵子发生过程中，卵子的细胞质中含有沉默的母体转录本，在受精后被激活，以响应发育线索。在初始转录开始之前，通过在特定时间在特定位置激活母体转录本的翻译来实现初始模式。少数RNA结合蛋白负责在卵子发生过程中沉默母体转录，并在受精后协调激活。在这里，我们定义了一个策略来筛选两个这样的蛋白的抑制剂，串联锌指蛋白MEX-5和POS-1。这些蛋白对线虫线虫的前后部细胞命运的决定至关重要，并与在调节人类炎症反应中起关键作用的Tristetraprolin有关。提出了一种识别、反筛选和验证小分子抑制剂的方法，该方法依赖于体外荧光分析和胚胎发育过程中的活体成像。通过这一筛选鉴定出的抑制性化合物可以作为“化学等位基因”，能够在时间和空间上剖析超出胚胎发生最早阶段的母体基因模式。这里描述的策略通常是有用的，可以适用于任何RNA结合蛋白，为筛选潜在的治疗靶向RNA结合蛋白的抑制剂打开了大门，包括曲曲普林，这可能导致类风湿性关节炎、牛皮癣和包括多发性硬化症在内的其他炎症性疾病的新疗法。这项建议描述了一种鉴定和验证与RNA结合的蛋白质的小化学抑制剂的方法。该策略将使用对蠕虫模型中胚胎干细胞分化至关重要的两个蛋白质靶点来完善。一旦优化，它可能被用于治疗相关的RNA结合蛋白，包括一种抑制炎症所需的相关蛋白。这项工作将与寻找治疗类风湿性关节炎、牛皮癣和其他炎症性疾病的新的、更好的治疗方法直接相关。此外，从这个项目中吸取的经验教训将有助于我们理解干细胞转化为所有类型的成体细胞的生物学过程。

DESCRIPTION (provided by applicant): A cohesive strategy is presented to identify small molecule effectors of maternal gene expression. During oogenesis, the cytoplasm of the egg is loaded with silenced maternal transcripts that are activated after fertilization in response to developmental cues. Initial patterning is achieved before nascent transcription begins by activating translation of maternal transcripts at specific times in defined locations. A handful of RNA-binding proteins are responsible for silencing maternal transcripts during oogenesis and coordinating activation after fertilization. Here, we define a strategy to screen for inhibitors of two such proteins, the tandem zinc finger proteins MEX-5 and POS-1. These proteins are critical for anterior and posterior cell fate determination in the nematode worm Caenorhabditis elegans, and are related to tristetraprolin, a factor critical in the regulation of the inflammation response in humans. A process for identifying, counter-screening, and validating small molecule inhibitors is presented that relies on both in vitro fluorescence assays and live imaging during embryogenesis. Inhibitory compounds identified through this screen could serve as "chemical alleles", enabling the temporal and spatial dissection of maternal gene patterning beyond the earliest points in embryogenesis. The strategy delineated here is generally useful and can be adapted to any RNA-binding protein, opening the door to screening for inhibitors of potentially therapeutic target RNA-binding proteins including tristetraprolin, which may lead to new therapies for rheumatoid arthritis, psoriasis, and other inflammatory diseases including multiple sclerosis. This proposal describes a method for identification and validation of small chemical inhibitors of proteins that bind to RNA. The strategy will be perfected using two protein targets that are critical to differentiation of embryonic stem cells in a worm model. Once optimized, it may be used for therapeutically relevant RNA-binding proteins, including a related protein that is required for deactivating inflammation. This work will have direct relevance to the search for new and better treatments for rheumatoid arthritis, psoriasis, and other inflammatory diseases. Moreover, the lessons learned from this project will contribute to our understanding of the biological processes that allow stem cells to change into all types of adult cells.