The Role of NMD in Embryonic Development

在胚胎发育的最早阶段，全能受精卵增殖和分化， 产生不同的细胞群体，最终形成成熟有机体的器官和组织。 这些早期事件决定了发育中的有机体的进程和成功。因此，至关重要的是， 调节这些早期发育转变的途径被阐明，以便诊断和理解 早期妊娠丢失，以及推进干细胞技术和不孕症治疗。大多数研究都有 集中在转录调控的作用上，这未能解释这样一个事实，即基因表达是 决定于信使核糖核酸衰变的速度与核糖核酸合成的速度一样多。在这个提案中，我们调查了 一种保守的、高选择性的RNA降解途径--无意义介导的RNA衰变(NMD)。 这一建议的首要假设是，NMD对早期胚胎发育至关重要，因为它 通过其调节关键RNA转录本的衰减率来影响特定的分化事件 特定于阶段的方式。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因子的全球丧失会导致早期胚胎死亡，并存在缺陷 在植入前和植入围术期表现明显。到目前为止，还没有研究检验其潜在的机制。 该领域另一个悬而未决的问题是如何监管NMD。理解这一点至关重要，因为NMD的转变 发育过程中的大小预计会引发数十个RNA稳定性的变化。一个 突破性的是我们最近发现了一种有效的NMD-UPF3A抑制因子。在大多数成人组织中检测不到， UPF3A在早期胚胎中高表达，小鼠UPF3A缺失可导致围产期死亡。 胚胎发育的着床阶段。这项提议的第一个目标是使用现有的鼠标KO模型 阐明NMD在发育过程中的作用--包括其被UPF3A抑制的必要性 活体早期胚胎的研究进展。为了准确定位NMD的作用机制，我们将使用单细胞 转录组分析，因为这将使我们能够(I)识别NMD作用的特定胚胎细胞亚群 和UPF3A，(Ii)定义在NMD作用的细胞子集中被NMD降解的mRNA谱，以及 (3)确定胚胎内随着发育进程发生的NMD活动的变化。第二个目标是 目的：阐明NMD在早期胚胎发育中重要作用的分子机制。利用 我们发现NMD对决定胚胎干细胞(ESCs)生殖层细胞命运至关重要，我们将使用 “模拟”和“拯救”方法来识别特定的mRNAs，NMD必须降解这些mRNAs才能驱动hESC 差异化决策。为了了解NMD监管如何影响这些事件，我们将研究 NMD抑制因子UPF3A在hESC分化中的发育和分子作用把这些放在一起 拟议的研究将首次定义对早期发育事件至关重要的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.