The Role of NMD in Embryonic Development

在胚胎发育的最早阶段，全能合子增殖和分化， 产生最终将形成成熟生物体的器官和组织的不同细胞群。 这些早期事件决定了发育有机体的进程和成功。因此，至关重要的是 阐明调节这些早期发育转变的途径，以诊断和理解 早期妊娠丢失，以及推进干细胞技术和不孕症治疗。大多数研究 集中在转录调控的作用，这不能解释基因表达是一个事实， mRNA的降解速率和RNA的合成速率同样决定了这一点。在本提案中，我们调查 一个保守的和高度选择性的RNA降解途径的作用-无义介导的RNA衰变（NMD）。 这个提议的首要假设是，NMD对早期胚胎发育至关重要，因为它 通过调节细胞中关键RNA转录物的衰变速率， 特定阶段的方式。作为支持，NMD降解编码发育调节因子和小鼠KO的RNA。 研究表明，几种NMD因子的全面缺失会导致早期胚胎死亡， 在植入前和植入期明显。到目前为止，还没有研究探讨了潜在的机制。 另一个悬而未决的问题是如何管理NMD。这一点很重要， 预测发育期间的变化幅度会触发RNA稳定性评分的改变。一 突破性进展是我们最近发现了NMD的有效阻遏物-UPF 3A。在大多数成人组织中检测不到， UPF 3A在早期胚胎中高度表达，小鼠中UPF 3A的缺失导致胚胎发育期间的死亡。 胚胎发育的着床阶段。本提案的第一个目的是使用现有的小鼠KO模型 阐明NMD的作用，包括UPF 3A抑制NMD的必要性， 早期胚胎在体内的发育。为了准确地说明NMD的作用机制，我们将用单细胞 转录组分析，因为这将使我们能够（i）确定NMD作用的特定胚胎细胞亚群 和UPF 3A，（ii）定义了NMD在其中起作用的细胞亚群中被NMD降解的mRNA库，和 (iii)确定随着发育的进行，胚胎内发生的NMD活动的变化。第二个目标是 阐明NMD在早期胚胎发生中重要作用的分子机制。利用 我们发现NMD对于决定胚胎干细胞（ESCs）中的胚层细胞命运至关重要，我们将使用 “模拟”和“拯救”方法来鉴定必须被NMD降解以驱动hESC的特异性mRNA 差异化决策。为了了解国家导弹防御系统的管制如何影响这些事件，我们将研究 NMD阻遏物UPF 3A在hESC分化中的发育和分子作用。综合这些 拟议的研究将首次定义一个对早期发育至关重要的RNA衰变网络， 类似于定义发育中转录网络的开创性研究。

During the earliest stages of embryonic development, the totipotent zygote proliferates and differentiates, generating diverse populations of cells that will ultimately form the organs and tissues of a mature organism. These early events dictate the course and success of the developing organism. Thus, it is critical that the pathways regulating these early developmental transitions are elucidated in order to diagnose and understand early pregnancy loss, as well as to advance stem cell technologies and infertility therapies. Most studies have concentrated on the role of transcriptional regulation, which fails to account for the fact that gene expression is dictated as much by the rate of mRNA decay as by the rate of RNA synthesis. In this proposal, we investigate the role of a conserved and highly selective RNA degradation pathway - Nonsense-Mediated RNA Decay (NMD). The overarching hypothesis of this proposal is that NMD is critical for early embryo development because it influences specific differentiation events through its ability to regulate the decay rate of key RNA transcripts in a stage-specific manner. In support, NMD degrades RNAs encoding developmental regulators and mouse KO studies have demonstrated that global loss of several NMD factors leads to early embryonic lethality, with defects evident during pre- and peri-implantation stages. To date, no studies have examined the underlying mechanism. Another outstanding issue in the field is how NMD is regulated. This is critical to understand, as shifts in NMD magnitude during development are predicted to trigger alterations in the stability of scores of RNAs. A breakthrough is our recent discovery of a potent repressor of NMD – UPF3A. Undetectable in most adult tissues, UPF3A is highly expressed in the early embryo, and loss of UPF3A in mice leads to lethality during the peri- implantation stage of embryo development. The first Aim of this proposal is to use existing mouse KO models to elucidate the roles of NMD—including the necessity of its repression by UPF3A—in the developmental progression of early embryos in vivo. To pinpoint NMD's mechanism of action, we will use single-cell transcriptome analysis, as this will allow us to (i) identify the specific embryonic cell subsets acted upon by NMD and UPF3A, (ii) define the repertoire of mRNAs degraded by NMD in the cell subsets in which NMD acts, and (iii) identify shifts in NMD activity that occur within the embryo as development proceeds. The second Aim is to elucidate the molecular mechanisms underlying NMD's essential roles in early embryogenesis. Leveraging our discovery that NMD is critical for dictating germ layer cell fate in embryonic stem cells (ESCs), we will use “mimic” and “rescue” approaches to identify the specific mRNAs that must be degraded by NMD to drive hESC differentiation decisions. To understand how NMD regulation influences these events, we will study the developmental and molecular roles of the NMD repressor, UPF3A, in hESC differentiation. Together these proposed studies will define—for the first time—a RNA decay network critical for early developmental events, akin to the pioneering studies defining transcriptional networks in development.