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

描述（由申请人提供）：提出了一种内聚策略来鉴定母体基因表达的小分子效应物。在卵子发生期间，卵子的细胞质装载有沉默的母体转录物，这些转录物在受精后响应于发育线索而被激活。在新生转录开始之前，通过在特定时间在定义的位置激活母体转录物的翻译来实现初始模式化。少数RNA结合蛋白负责在卵子发生期间沉默母体转录物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质，串联锌指蛋白MEX-5和POS-1的抑制剂。这些蛋白质对于线虫秀丽隐杆线虫中的前细胞和后细胞命运决定至关重要，并且与tristetraprolin相关，tristetraprolin是调节人类炎症反应的关键因子。提出了一种用于鉴定、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测定和胚胎发生期间的活体成像。通过这种筛选确定的抑制性化合物可以作为“化学等位基因”，使时间和空间解剖母体基因模式超越胚胎发生的最早点。这里描述的策略通常是有用的，并且可以适用于任何RNA结合蛋白，为筛选潜在治疗靶RNA结合蛋白（包括tristetraprolin）的抑制剂打开了大门，这可能导致类风湿性关节炎，银屑病和其他炎症性疾病（包括多发性硬化症）的新疗法。该提案描述了一种用于鉴定和验证与RNA结合的蛋白质的小化学抑制剂的方法。该策略将使用两种对蠕虫模型中胚胎干细胞分化至关重要的蛋白质靶点来完善。一旦优化，它可以用于治疗相关的RNA结合蛋白，包括灭活炎症所需的相关蛋白。这项工作将直接关系到寻找新的和更好的治疗类风湿性关节炎，牛皮癣和其他炎症性疾病。此外，从这个项目中吸取的经验教训将有助于我们理解允许干细胞转变为所有类型的成体细胞的生物过程。

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.