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

秀丽隐杆线虫胚胎发生中母体基因表达的小分子效应器

基本信息

批准号：
7680768
负责人：
Sean Patrick Ryder
金额：
$ 4.09万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-09-30 至 2009-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7680768
关键词：
3&apos Untranslated Regions Adult Alleles Animals Anterior Binding Biological Assay Biological Process Caenorhabditis elegans Cells Chemicals Condition Cues Cytoplasm Defect Development Disease Dissection Electrophoretic Mobility Shift Assay Embryo Embryology Embryonic Development Family Fertilization Fluorescence Fluorescence Microscopy Fluorescence Polarization Gene Expression Genes Genetic Transcription Goals Green Fluorescent Proteins Human Image In Vitro Indium Inflammation Inflammatory Label Lead Learning Libraries Life Location Messenger RNA Methods Modeling Monitor Multiple Sclerosis Nematoda Nucleotides Numbers Oogenesis Pattern Phenotype Post-Transcriptional Regulation Process Protein Binding Proteins Protocols documentation Psoriasis RNA RNA Binding RNA-Binding Proteins Rate Regulation Rheumatoid Arthritis Score Screening procedure Series Somatic Cell Specificity Staging Standards of Weights and Measures Stem cells TIS11 protein Testing Time Tissue Differentiation Transcript Transgenic Organisms Translations Validation Work Zinc Fingers blastomere structure chemical genetics egg embryo stage 2 embryonic protein embryonic stem cell genetic analysis high throughput screening human RBM5 protein in vitro Assay inhibitor/antagonist killings medical schools member pleiotropism protein expression research study response segregation small molecule therapeutic target tool

项目摘要

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.

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子。在卵子发生过程中，卵子的细胞质中充满了沉默的母体转录物，这些转录物在受精后响应发育线索而被激活。在新生转录开始之前，通过在指定位置的特定时间激活母体转录本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过程中沉默母体转录本并协调受精后的激活。在这里，我们定义了一种筛选两种此类蛋白质（串联锌指蛋白 MEX-5 和 POS-1）抑制剂的策略。这些蛋白质对于线虫秀丽隐杆线虫的前部和后部细胞命运决定至关重要，并且与三四脯氨酸有关，三四脯氨酸是调节人类炎症反应的关键因素。提出了一种识别、反筛选和验证小分子抑制剂的过程，该过程依赖于胚胎发生过程中的体外荧光测定和实时成像。通过该筛选鉴定的抑制性化合物可以充当“化学等位基因”，从而能够在胚胎发生的最早点之外对母体基因模式进行时间和空间解剖。这里描述的策略通常是有用的，可以适应任何 RNA 结合蛋白，为筛选潜在治疗靶标 RNA 结合蛋白（包括三四脯氨酸）的抑制剂打开了大门，这可能会导致类风湿性关节炎、牛皮癣和其他疾病的新疗法。炎症性疾病，包括多发性硬化症。该提案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。该策略将通过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标来完善。一旦优化，它可用于治疗相关的 RNA 结合蛋白，包括灭活炎症所需的相关蛋白。这项工作将与寻找新的、更好的治疗类风湿关节炎、牛皮癣和其他炎症性疾病的方法直接相关。此外，从该项目中汲取的经验教训将有助于我们了解干细胞转变为所有类型成体细胞的生物过程。