Post-transcriptional gene regulation by the exon junction complex

项目摘要 无义突变对细胞和有机体的适合性和生存构成了严重的挑战。压制 携带这种无义突变的mrna，所有真核生物都有一条保守的mrna监控途径。 称为无意义介导的信使核糖核酸衰变(NMD)。NMD也是一种重要的转录后调节因子 形成干细胞维持、神经发生、生殖细胞发育等过程的正常mRNA 和抗病毒反应。在所有真核生物中，NMD由UPF1、UPF2和UPF3三种UPF蛋白调控。在……里面 在多细胞生物中，NMD也受到一个保守的多蛋白外显子连接复合体(EJC)的调节，该复合体 与mRNA外显子-外显子连接的上游结合。在转换过程中，如果至少有一个EJC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的机制及其受EJC的调控对改善人类健康至关重要，因为EJC和 NMD蛋白会导致发育缺陷、智力残疾和智力低下。首要目标是 这项研究计划的目的是了解EJC/UPF在组成和功能上的显著变化 机械调节NMD来决定细胞功能和动物细胞的命运。为了实现这一目标，我们正在使用 基因、基因组、分子、生化和细胞方法在培养的人类细胞和 在斑马鱼胚胎中追求四个主要方向。(1)我们将确定EJC中的切换机制 我们最近发现并定义了不同的EJC成分在基因表达中的作用。(2) 我们最近发现，哺乳动物UPF3并列基因及其与EJC的相互作用对NMD不是必需的 挑战真核生物数十年来依赖于EJC的NMD模型。我们将应用新的基因组技术 该探针在体内的核糖体功能以确定UPF3和其他UPF蛋白在早产中的作用 数百个人类mRNA上的复杂组装和活性。我们还将确定以下因素和特点 管理终端复合体和EJC之间的信令。(3)我们和其他人之前已经证明 通常在RNAs上的意外位置检测到JEC。通过利用EJC回收的新步骤，我们 已经发现，我们将定义在这些意想不到的位置上的EJB的组装机制和功能。(4) 我们已经开发出缺少一种EJC或其NMD适配蛋白的斑马鱼突变体，这将被用于 确定这些因素在运动神经元和肌肉过程中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机制及其如何监管后 转录基因调控以控制细胞功能和生物发育。这一进展还将 提升我们针对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.