Post-transcriptional gene regulation by the exon junction complex

项目摘要 无义突变对细胞和生物体的适应性和生存构成了严重挑战。抑制 携带这种无义突变的mRNA，所有真核生物都具有保守的mRNA监视途径 无义介导的mRNA衰变（NMD）NMD也是一种重要的转录后调节因子， 正常的mRNA，塑造过程，如干细胞维持，神经发生，生殖细胞发育 和抗病毒反应。在所有真核生物中，NMD由三种UPF蛋白（UPF 1、UPF 2和UPF 3）控制。在 在多细胞生物中，NMD也受保守的多蛋白外显子连接复合物（EJC）调节， 结合mRNA外显子-外显子连接的上游。在翻译过程中，如果至少有一个EJC仍然存在 在终止核糖体的下游，它可以发出提前终止的信号并触发NMD。理解 NMD机制及其通过EJC的调节对于改善人类健康至关重要，因为EJC的突变和 NMD蛋白导致发育缺陷、智力残疾和智力迟钝。总体目标 本研究计划的目的是了解EJC/UPF的组成和功能的显着变化如何影响EJC/UPF的功能。 机械调节NMD以决定动物细胞中的细胞功能和命运。为了实现这一目标，我们正在使用 在培养的人类细胞中结合遗传、基因组、分子、生物化学和细胞方法， 在斑马鱼胚胎中，有四个主要方向。(1)我们将确定EJC中开关的机制 我们最近发现并定义了不同的EJC组合物在基因表达中的作用。（二） 我们最近发现哺乳动物UPF 3旁系同源物及其与EJC的相互作用对于NMD是非必需的 挑战了真核生物中依赖EJC的NMD的几十年模型。我们将应用新的基因组技术 其探测体内核糖体功能以鉴定UPF 3和其它UPF蛋白在过早终止中的作用 复杂的组装和数百种人类mRNA的活性。我们还将确定的因素和特点， 控制终止复合物和EJC之间的信号传导。(3)我们和其他人之前已经证明， EJC通常在RNA上的意外位置检测到。通过利用EJC回收的新步骤， 已经发现，我们将定义EJCs在这些意想不到的网站的组装机制和功能。（四） 我们已经开发了斑马鱼突变体，它们缺乏EJC或其NMD衔接蛋白之一，这将用于 以确定运动神经元和肌肉中由这些因素控制的遗传和细胞过程， 发展总体而言，我们的工作将促进对NMD机制的了解，以及它们如何调节后导弹防御系统。 转录基因调控来控制细胞功能和生物体发育。这一进展也将 提高我们针对NMD进行治疗的能力。

Project Summary Nonsense mutations pose a serious challenge to fitness and survival of cells and organisms. To suppress mRNAs carrying such nonsense mutations, all eukaryotes possess a conserved mRNA surveillance pathway called Nonsense-Mediated mRNA Decay (NMD). NMD is also an essential post-transcriptional regulator of normal mRNAs that shapes processes such as stem cell maintenance, neurogenesis, germ cell development and anti-viral response. In all eukaryotes, NMD is governed by three UPF proteins, UPF1, UPF2 and UPF3. In multicellular organisms, NMD is also regulated by a conserved multi-protein exon junction complex (EJC), which binds upstream of mRNA exon-exon junctions. During translation, if at least one EJC remains present downstream of a terminating ribosome, it can signal premature termination and trigger NMD. Understanding NMD mechanism and its regulation by EJC is crucial for betterment of human health as mutations in EJC and NMD proteins cause developmental defects, intellectual disability and mental retardation. The overarching goal of this research program is to understand how the remarkable variation in composition and function of EJC/UPF machinery regulates NMD to dictate cellular function and fate in animal cells. To achieve this goal, we are using a combination of genetic, genomic, molecular, biochemical and cellular approaches in cultured human cells and in zebrafish embryos to pursue four main directions. (1) We will identify the mechanism of a switch in EJC composition that we recently discovered and define the role of distinct EJC compositions in gene expression. (2) Our recent discovery that mammalian UPF3 paralogs and their interaction with EJC are non-essential for NMD challenges a decades old model of EJC-dependent NMD in eukaryotes. We will apply new genomic technologies that probe in vivo ribosome function to identify the role of UPF3 and other UPF proteins in premature termination complex assembly and activity on hundreds of human mRNAs. We will also identify the factors and features that govern signaling between the termination complex and the EJC. (3) We and others have previously shown that EJCs are often detected at unexpected locations on RNAs. By exploiting a new step in EJC recycling that we have uncovered, we will define the assembly mechanisms and functions of EJCs at such unexpected sites. (4) We have developed zebrafish mutants that lack one of the EJC or its NMD adapter proteins, which will be used to identify the genetic and cellular processes controlled by these factors during motor neuron and muscle development. Overall, our work will advance the knowledge of NMD mechanisms and how they regulate post- transcriptional gene regulation to control cellular function and organismal development. This progress will also elevate our ability to target NMD for therapeutics.