Region-specific activation of oskar mRNA translation by inhibition of Bruno-mediated repression.
Region-specific activation of oskar mRNA translation by inhibition of Bruno-mediated repression.
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DOI：
10.1371/journal.pgen.1004992
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发表时间：
2015
期刊：
影响因子：
4.5
通讯作者：
Macdonald PM
中科院分区：
文献类型：
--
作者：
Kim G;Pai CI;Sato K;Person MD;Nakamura A;Macdonald PM
翻译抑制、信使核糖核酸定位和翻译激活的复杂程序确保Oskar(OSK)蛋白只在果蝇卵母细胞的后极积累。不适当的OSK表达会破坏胚胎的轴向模式，并是致命的。翻译抑制的一个关键因素是Bruno(Bru)，它与OSK mRNA3‘UTR中的调控元件结合。在OSK信使核糖核酸定位后，Bru的抑制作用必须得到缓解。在这里，我们描述了一种体内检测系统，用于监测Bru依赖的抑制的空间模式，与OSK调控的全部复杂性分开。这种分析揭示了一种翻译激活的形式-区域特异性激活-在卵母细胞中发挥区域性作用，并不是机械地与mRNA定位耦合，而是通过抑制Bru的抑制来发挥作用。我们还证明了Bru二聚化并确定了破坏这种相互作用的突变，以测试其在体内的作用。二聚化的丧失并不会扰乱压制，正如从现有的压制机制模型中可以预料到的那样。然而，二聚化的丧失确实损害了翻译的区域激活，这表明二聚化可能抑制而不是促进抑制。我们的工作为本地化的mRNAs如何在翻译上变得活跃的问题提供了新的见解，表明OSK mRNA的抑制是由一种独立于mRNA本地化的机制局部失活的。蛋白质通常通过mRNAs的定位被浓缩到细胞内的特定区域。这种现象发挥着多种作用，既可以将特定细胞过程中涉及的因素聚集在一起以提高其效率，也可以限制如果部署在不适当的位置可能会造成伤害的蛋白质。在后一种情况下，翻译抑制在mRNA本地化之前阻止表达，必须有激活机制来抑制或覆盖抑制。MRNA定位和翻译过程是如何协调的还不是很清楚，部分原因是为了在体外研究机制而准备的细胞提取物不保留完整细胞中存在的空间信息。我们开发了一种体内试验来监测果蝇卵母细胞的翻译模式，其中几个模式决定因素必须定位到特定的区域。使用这一检测，我们表明Bruno蛋白对翻译的抑制是被抑制的，我们可以直观地看到这种抑制发生在卵子发生的时间和地点。区域激活不仅发生在mRNA定位的位置，而且更广泛地以分级的方式发生，并且它不需要在信使核糖核酸中有激活元件。我们还表明Bruno是二聚化的，并且二聚化对于翻译激活是重要的。
A complex program of translational repression, mRNA localization, and translational activation ensures that Oskar (Osk) protein accumulates only at the posterior pole of the Drosophila oocyte. Inappropriate expression of Osk disrupts embryonic axial patterning, and is lethal. A key factor in translational repression is Bruno (Bru), which binds to regulatory elements in the osk mRNA 3′ UTR. After posterior localization of osk mRNA, repression by Bru must be alleviated. Here we describe an in vivo assay system to monitor the spatial pattern of Bru-dependent repression, separate from the full complexity of osk regulation. This assay reveals a form of translational activation—region-specific activation—which acts regionally in the oocyte, is not mechanistically coupled to mRNA localization, and functions by inhibiting repression by Bru. We also show that Bru dimerizes and identify mutations that disrupt this interaction to test its role in vivo. Loss of dimerization does not disrupt repression, as might have been expected from an existing model for the mechanism of repression. However, loss of dimerization does impair regional activation of translation, suggesting that dimerization may constrain, not promote, repression. Our work provides new insight into the question of how localized mRNAs become translationally active, showing that repression of osk mRNA is locally inactivated by a mechanism acting independent of mRNA localization. Proteins are often enriched to specific regions within cells via localization of mRNAs. This phenomenon serves a variety of roles, both bringing together factors involved in particular cellular processes to enhance their efficiency, and in restricting proteins that could do harm if deployed at inappropriate positions. In the latter situation, translational repression prevents expression before mRNA localization, and there must be activation mechanisms to inhibit or override repression. How the processes of mRNA localization and translation are coordinated is not well understood, in part because cellular extracts prepared to study mechanisms in vitro do not retain the spatial information present in the intact cell. We developed an in vivo assay to monitor the pattern of translation in the Drosophila oocyte, where several patterning determinants must be localized to specific regions. Using this assay, we showed that repression of translation by the Bruno protein is inhibited, and we could visualize when and where this occurs during oogenesis. Regional activation occurs not only at the site of mRNA localization, but more broadly in a graded fashion, and it does not require an activation element in the mRNA. We also show that Bruno dimerizes, and that dimerization is important for translational activation.
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