Novel Regulatory Mechanisms of Drosophila Pumilio

项目总结 我们研究的长期目标是确定基因表达是如何在后 转录水平。控制信使RNA的翻译和降解是重要的基础 生物过程包括发育、生育和神经功能。拟议的工作重点是 典型的mRNA调节因子小果蝇(Pum)，属于RNA结合蛋白家族 它们在整个尤卡里亚都是保守的。PUM结合了一组广泛的mRNA，包括那些编码 关键发育形态因子。一旦与mRNA结合，Pum就会抑制编码的 蛋白。我们发现Pum加速了靶mRNA的衰退，拟议的研究试图发现 Pum介导的mRNA降解机制。我们开发了新的方法来测量Pum的活性 果蝇细胞，并发现了多个独特的抑制结构域，有效地抑制了目标mRNA。在……里面 在前期工作中，我们确定了Pum抑制所必需的关键辅助抑制因子。在第一个目标中，我们 测量抑制域对蛋白质表达和mRNA降解的影响并询问 两类信使核糖核酸衰变因子在Pum抑制结构域抑制中的作用。我们 询问共抑制物与每个Pum抑制结构域的物理相互作用并测量 它们被招募到受PUM调控的靶向RNA。在第二个目标中，我们调查每个Pum的作用 胚胎发育中的抑制域与基因表达的适当时空调控 在早期发育过程中。这项研究将揭示Pum抑制的新机制。由此产生的 这些发现有望广泛地增强我们对转录后调控的理解，特别是 提高在发育、生育、干细胞增殖和神经系统方面的基因调控知识。

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.