Novel Regulatory Mechanisms of Drosophila Pumilio

项目摘要 我们研究的长期目标是确定基因表达是如何调节的， 转录水平。信使RNA（mRNA）翻译和降解的控制是重要的 包括发育、生育和神经功能在内的生物过程。拟议工作的重点是 原型mRNA调节子，果蝇Pumilio（Pum），属于RNA结合蛋白家族 在整个真核生物中都保存着。Pum结合了大量的mRNA，包括那些编码 关键的发育形态因子。在与mRNA结合后，Pum抑制编码的 蛋白我们发现Pum加速了靶mRNA的衰变，并且拟议的研究旨在发现 Pump介导的mRNA降解机制。我们开发了新的测定方法来测量Pum活性， 果蝇细胞，发现了多个独特的抑制结构域，有效地抑制靶mRNA。在 在初步工作中，我们确定了Pum抑制所必需的关键辅抑制因子。第一个目标，我们 测量阻遏结构域对蛋白质表达和mRNA降解的影响， 两类mRNA衰减因子在每个Pum阻遏结构域阻遏中的功能作用。我们 询问辅阻遏物与每个Pum阻遏结构域的物理相互作用，并测量 将其募集到Pump调节的靶mRNA。在第二个目标中，我们研究了每个Pum的作用， 胚胎发育中的阻遏结构域与基因表达的时空调控 在早期发展中。这项研究将揭示Pum抑制的新机制。所得 这些发现有望广泛增强我们对转录后控制的理解， 提高对发育、生育、干细胞增殖和神经系统基因调控的认识。

PROJECT SUMMARY The long term goal of our research is to determine 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, Drosophila Pumilio (Pum), which belongs to a family of RNA-binding proteins that are conserved throughout eukarya. Pum binds an extensive group of mRNAs including those that encode key developmental morphogens. Upon binding to an mRNA, Pum represses expression of the encoded protein. We find that Pum accelerates decay of the target mRNA and the proposed research seeks to discover the mechanism of Pum-mediated mRNA degradation. We developed novel assays to measure Pum activity in Drosophila cells and discovered multiple unique Repression Domains that potently repress target mRNAs. In preliminary work, we identified key co-repressors that are necessary for Pum repression. In the first aim, we measure the impact of the Repression Domains on protein expression and mRNA degradation and interrogate the functional roles of two classes of mRNA decay factors in repression by each Pum Repression Domain. We interrogate the physical interactions of the co-repressors with each Pum Repression Domain and measure their recruitment to Pum-regulated target mRNAs. In the second aim, we investigate the role of each Pum Repression Domain in embryonic development and proper spatial and temporal control of gene expression during early development. This research will reveal novel mechanisms of Pum repression. 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.