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Mechanistic analysis of nanos mRNA translational regulation.

机译：纳米mRNA的翻译调控机制分析。

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摘要

Nanos is critical for patterning the posterior half of the embryo during early development. Loss of nanos leads to embryos that fail to develop abdominal segments, while overexpression or mislocalization of nanos leads to embryos that develop additional abdominal fates at the expense of anterior fates. A gradient of Nanos protein is synthesized from maternally deposited nanos mRNA that is localized to the posterior of the embryo, and localization of nanos is necessary to activate its translation and produce this gradient. However, only 4% of the total pool of nanos mRNA is actually localized and it is essential that the unlocalized mRNA be translationally repressed for proper development to occur. Translational repression of nanos is conferred by the translational control element in the nanos 3'UTR. One key component of the embryonic translational repression machinery, the protein Smaug, has been identified, and recently a second protein, Glorund, has been shown to repress nanos translation during oogenesis. Smaug has been proposed to repress nanos by blocking translational initiation. The mechanism by which Glorund acts to repress translation in the ovary is not known. Polysome sedimentation gradients have shown that translationally repressed nanos RNA is associated with polysomes, suggesting that at least some aspects of translational control may occur post-initiation.;To determine whether repression acts at initiation, post-initiation, or both, we have developed a Drosophila ovarian extract that faithfully recapitulates nanos translational repression and have used this extract to further characterize the mode of repression in both the embryo and the ovary. Our results indicate that while translational repression of nanos mRNA in the embryo extract occurs entirely at translational initiation, translational repression in the ovarian extract operates by two modes of repression. One of these acts at initiation and has a strong dependence on the presence of a poly(A) tail, while the other acts post-initiation in a cap-independent manner.;In a second approach to studying translational control of nanos , we have examined the regulation of nanos at the neuromuscular junction. During embryonic development in Drosophila, Nanos acts together with the RNA-binding protein Pumilio to direct posterior patterning. Nanos and Pumilio also function to regulate synaptic growth and plasticity at the neuromuscular junction, but in that context appear to act in opposition to each other. Postsynaptic Pumilio negatively regulates the expression of several proteins, including eIF4E, the Glutamate RIIA receptor subunit, and Nanos itself. In contrast, Nanos positively regulates Glutamate RIIA translation. We have examined whether the observed regulation of Glutamate RIIA and nanos by Pumilio could result from a direct interaction using a series of gel mobility shift assays. Our results demonstrate that Pumilio protein binds selectively and directly to the Glutamate RIIA 3'UTR as well as to the nanos 3'UTR, and identifies a novel binding target for Pumilio.
机译：纳米对于在早期发育中形成胚胎后半部分至关重要。纳米的损失导致胚胎无法发育腹节，而纳米的过度表达或定位错误导致胚胎产生更多的腹部命运，但牺牲了前路命运。 Nanos蛋白的梯度是从母体沉积的nanos mRNA合成的，该mRNA定位在胚胎的后部，而nanos的定位对于激活其翻译并产生该梯度是必需的。但是，实际上只有4％的nanos mRNA池被定位，必须对未定位的mRNA进行翻译抑制，以使其正常发育。纳米的平移抑制是由纳米3'UTR中的翻译控制元件赋予的。胚胎翻译抑制机制的一个重要组成部分，即蛋白质Smaug，已被鉴定，最近，第二种蛋白质Glorund被证明在卵子发生过程中抑制纳米翻译。已经提出了Smaug通过阻断翻译起始来抑制纳米。 Glorund抑制卵巢翻译的机制尚不清楚。多核糖体沉淀梯度表明，翻译抑制的纳米RNA与多核糖体相关，这表明翻译控制的至少某些方面可能在启动后发生;为了确定抑制在启动，启动后还是同时发生，我们开发了一种果蝇卵巢提取物忠实地概括了纳米翻译抑制，并已使用该提取物进一步表征了胚胎和卵巢中的抑制模式。我们的结果表明，虽然胚胎提取物中的nanos mRNA的翻译抑制完全发生在翻译起始时，但卵巢提取物中的翻译抑制却通过两种抑制模式起作用。其中一种在引发时起作用，并且强烈依赖于poly（A）尾部的存在，而另一种以与帽不相关的方式在引发后起作用。;在研究纳米的翻译控制的第二种方法中，研究了神经肌肉接头处纳米的调节。在果蝇的胚胎发育过程中，Nanos与RNA结合蛋白Pumilio共同作用，以指导后部模式。 Nanos和Pumilio也起着调节神经肌肉连接处突触生长和可塑性的作用，但是在这种情况下似乎彼此相反。突触后小肠负性调节几种蛋白质的表达，包括eIF4E，谷氨酸RIIA受体亚基和Nanos本身。相反，Nanos积极调节谷氨酸RIIA的翻译。我们已经检查了通过Pumilio观察到的谷氨酸RIIA和纳米颗粒的调节是否可以由使用一系列凝胶迁移率移动分析的直接相互作用导致。我们的结果表明，Pumilio蛋白选择性地直接结合到谷氨酸RIIA 3'UTR以及纳米3'UTR上，并确定了Pumilio的新型结合靶标。

著录项

  • 作者

    Andrews, Shane.;

  • 作者单位

    Princeton University.;

  • 授予单位 Princeton University.;
  • 学科 Biology Molecular.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 98 p.
  • 总页数 98
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

  • 入库时间 2022-08-17 11:38:19

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