Post-transcriptional gene regulation by the exon junction complex

外显子连接复合物的转录后基因调控

• 批准号: 10623701
• 负责人: Guramrit Singh
• 金额: $ 37.91万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2028-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10623701
• 关键词: Adaptor Signaling Protein; Animals; Antiviral Response; Binding; Biochemical; Cell Maintenance; Cell Survival; Cell physiology; Cells; Complex; Defect; Development; Embryo; Eukaryota; Eukaryotic Cell; Exons; Gene Expression; Genetic; Genetic Processes; Genomics; Germ Cells; Goals; Health; Human; Intellectual functioning disability; Knowledge; Lead; Location; Mediating; Mental Retardation; Messenger RNA; Modeling; Molecular; Muscle; Muscle Development; Mutation; Neurons; Nonsense Mutation; Organism; Pathway interactions; Post-Transcriptional Regulation; Process; Proteins; RNA; Recycling; Regulation; Research; Ribosomes; Role; Shapes; Signal Transduction; Site; Technology; Therapeutic; Tissues; Translations; Variant; Work; Zebrafish; autism spectrum disorder; fitness; human disease; in vivo; mRNA Decay; mRNA Surveillance; motor neuron development; mutant; neurogenesis; novel therapeutics; paralogous gene; posttranscriptional; premature; programs; stem cells

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.

项目摘要 废话突变对细胞和生物的适应性和存活构成了严重的挑战。抑制 携带这种胡说突变的mRNA，所有真核生物都具有保守的mRNA监视途径 称为废话介导的mRNA衰变（NMD）。 NMD也是首要的转录后调节器 正常的mRNA，塑造了干细胞维持，神经发生，生殖细胞发育等过程 和抗病毒反应。在所有真核生物中，NMD均由三种UPF蛋白（UPF1，UPF2和UPF3）支配。在 多细胞生物，NMD也受保守的多蛋白外显子连接络合物（EJC）的调节，该复合物（EJC） 绑定mRNA外显子 - 外观连接处的上游。在翻译过程中，如果至少有一个EJC存在 终止核糖体的下游，它可以发出过早终止的信号并触发NMD。理解 NMD机制及其对EJC的调节对于改善人类健康作为EJC和EJC的突变至关重要 NMD蛋白会导致发育缺陷，智力残疾和智力低下。总体目标 该研究计划的内容是了解EJC/UPF组成和功能的显着变化 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 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中开关的机制 我们最近发现并定义了不同EJC组成在基因表达中的作用。 （2） 我们最近发现的是，哺乳动物UPF3旁系同源物及其与EJC的互动对于NMD不必要 挑战真核生物中EJC依赖性NMD的数十年模型。我们将应用新的基因组技术 该探测体内核糖体功能，以识别UPF3和其他UPF蛋白在过早终止中的作用 数百个人类mRNA的复杂组装和活动。我们还将确定因素和特征 控制终止复合物与EJC之间的信号传导。 （3）我们和其他人以前已经表明 经常在RNA的意外位置检测到EJC。通过利用EJC回收的新步骤，我们 已经发现，我们将定义EJC在此类意外站点上的组装机制和功能。 （4） 我们已经开发了缺乏EJC或其NMD适配器蛋白之一的斑马鱼突变体，将被使用 确定运动神经元和肌肉期间这些因素控制的遗传和细胞过程 发展。总体而言，我们的工作将提高NMD机制的了解以及它们如何调节后 转录基因调节以控制细胞功能和生物发育。这个进展也将 提高了我们针对NMD治疗剂的能力。