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.

项目概要 无义突变对细胞和生物体的适应性和生存提出了严峻的挑战。压制 携带此类无义突变的 mRNA，所有真核生物都拥有保守的 mRNA 监视途径 称为无义介导的 mRNA 衰变 (NMD)。 NMD 也是重要的转录后调节因子 塑造干细胞维持、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 受三种 UPF 蛋白 UPF1、UPF2 和 UPF3 控制。在 在多细胞生物中，NMD 还受到保守的多蛋白外显子连接复合物 (EJC) 的调节，该复合物 结合 mRNA 外显子-外显子连接的上游。翻译过程中，如果至少有一个 EJC 仍然存在 它位于终止核糖体的下游，可以发出提前终止信号并触发 NMD。理解 NMD 机制及其 EJC 的调节对于改善人类健康至关重要，因为 EJC 和 NMD 的突变 NMD 蛋白会导致发育缺陷、智力障碍和精神发育迟滞。总体目标 该研究计划的目的是了解 EJC/UPF 的组成和功能的显着变化是如何发生的 机器调节 NMD 来决定动物细胞的细胞功能和命运。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、生化和细胞方法 在斑马鱼胚胎中追求四个主要方向。 (1)我们将识别EJC中的切换机制 我们最近发现了这些成分，并定义了不同 EJC 成分在基因表达中的作用。 (2) 我们最近发现哺乳动物 UPF3 旁系同源物及其与 EJC 的相互作用对于 NMD 不是必需的 挑战了真核生物中几十年前依赖 EJC 的 NMD 模型。我们将应用新的基因组技术 探测体内核糖体功能以确定 UPF3 和其他 UPF 蛋白在过早终止中的作用 数百种人类 mRNA 的复杂组装和活性。我们还将确定影响因素和特征 控制终止复合体和 EJC 之间的信号传导。 (3) 我们和其他人之前已经证明 EJC 经常在 RNA 上意想不到的位置被检测到。通过利用 EJC 回收的新步骤，我们 我们发现，我们将在这些意想不到的地点定义 EJC 的组装机制和功能。 (4) 我们开发了缺乏 EJC 或其 NMD 接头蛋白之一的斑马鱼突变体，该突变体将用于 识别运动神经元和肌肉过程中受这些因素控制的遗传和细胞过程 发展。总体而言，我们的工作将增进对 NMD 机制及其如何监管后的认识。 转录基因调控控制细胞功能和有机体发育。这一进展也将 提高我们针对 NMD 进行治疗的能力。