Request to Transfer R01GM105707-03 Novel regulatory mechanisms of Drosophila Pumilio and Nanos

描述(由申请人提供)：这项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。控制信使RNA的翻译和降解是包括发育、生育和神经功能在内的重要生物学过程的基础。拟议的工作重点是原型mRNA调节因子Pum(Pum)。PUM是真核RNA结合蛋白PUF家族中的一员。PUM结合了一组广泛的信使RNA，包括那些编码细胞周期蛋白和关键发育形态原的信使RNA。与信使核糖核酸结合后，Pum抑制编码蛋白的表达。这项拟议的研究旨在发现Pum抑制的机制，并确定Pum活性是如何调节的。我们开发了新的方法来测量果蝇细胞中Pum的活性，并发现了多个有效抑制蛋白质表达的新结构域。Pum抑制结构域的抑制活性不依赖于先前已发现的辅助因子，表明了新的抑制机制(S)。在第一个目标中，我们测量了抑制结构域对翻译和mRNA降解的影响。我们试图发现他们活动所必需的辅助因素。我们发现了一种控制Pum活性的自动调节开关，并可能受到磷酸化的调节。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我调节开关的机制。在第三个目的中，我们探索了锌指蛋白纳米是如何调节Pum的抑制的。Nanos与Pum相互作用，增强了抑制作用。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mRNAs的收集。这项研究将确定Pum抑制的新机制，并阐明该活性如何受到蛋白质修饰和与Nanos的组合作用的调节。由此产生的发现有望广泛地增强我们对转录后调控的理解，特别是提高对发育、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.