Novel regulatory mechanisms of Drosophila Pumilio and Nanos

描述（由申请人提供）：本研究的长期目标是揭示基因表达是如何在转录后水平上调节的。信使RNA（mRNA）的翻译和降解的控制是重要的生物过程，包括发育，生育和神经功能的基础。拟议的工作集中在原型mRNA调节子Pumilio（Pum）上。Pum是真核生物RNA结合蛋白PUF家族的成员。Pum结合了大量的信使RNA，包括那些编码细胞周期蛋白和关键发育形态因子的信使RNA。在与mRNA结合后，Pum抑制编码蛋白的表达。这项研究旨在发现Pum抑制的机制，并确定Pum活性是如何调节的。我们开发了新的检测方法来测量果蝇细胞中的Pum活性，并发现了多个有效抑制蛋白质表达的新结构域。Pum阻遏结构域的阻遏活性不依赖于先前鉴定的辅因子，表明新的阻遏机制。在第一个目标中，我们测量阻遏结构域对翻译和mRNA降解的影响。我们试图发现其活动所必需的辅助因子。我们发现了一个自动调节开关，控制Pum的活动，并可能通过磷酸化调节。在第二个目标中，我们使用功能和蛋白质相互作用分析来剖析这种自动调节开关的机制。在第三个目标中，我们探索锌指蛋白Nanos如何调节Pum的阻遏。Nanos与Pum相互作用并增强抑制作用。我们认为Nanos增强了Pum与mRNA的相互作用，实际上改变了Pum抑制的mRNA的收集。这项研究将确定Pum抑制的新机制，并阐明该活性如何响应蛋白质修饰和与Nanos的组合作用而受到调节。由此产生的发现有望广泛增强我们对转录后控制的理解，并特别提高发育，生育，干细胞增殖和神经系统中基因调控的知识。

DESCRIPTION (provided by applicant): The long term goal of this research is to reveal how gene expression is regulated at the post-transcriptional level. Control of messenger RNA (mRNA) translation and degradation underlies important biological processes including development, fertility and neurological functions. The proposed work focuses on the archetypal mRNA regulator, Pumilio (Pum). Pum is a member of the PUF family of eukaryotic RNA binding proteins. Pum binds an extensive group of messenger RNAs including those that encode cell cycle proteins and key developmental morphogens. Upon binding to an mRNA, Pum represses expression of the encoded protein. The proposed research seeks to discover the mechanism of Pum repression and determine how Pum activity is modulated. We developed novel assays to measure Pum activity in Drosophila cells and discovered multiple new domains that potently repress protein expression. The repressive activity of the Pum repression domains do not rely on previously identified cofactors, indicating novel repression mechanism(s). In the first aim, we measure the impact of the repression domains on translation and mRNA degradation. We seek to discover cofactors necessary for their activity. We discovered an autoregulatory switch that controls Pum activity and may be modulated by phosphorylation. In the second aim, we dissect the mechanism of this autoregulatory switch using functional and protein interaction assays. In the third aim, we explore how the Zn finger protein Nanos regulates repression by Pum. Nanos interacts with Pum and enhances repression. We propose that Nanos enhances interaction of Pum with mRNA and, in effect, changes the collection of mRNAs repressed by Pum. This research will determine novel mechanisms of Pum repression and illuminate how that activity is regulated in response to protein modifications and combinatorial action with Nanos. The resulting discoveries are expected to broadly enhance our understanding of post-transcriptional control and specifically improve knowledge of gene regulation in development, fertility, stem cell proliferation, and the nervous system.