Formation of the repressed nanos mRNP in the early Drosophila embryo

母体RNA是由母亲提供给发育中的卵母细胞的RNA。这类RNA控制早期发育，并受转录后机制的调节。其中一个这样的RNA是Nanos信使核糖核酸，它编码早期果蝇胚胎后期发育的决定因素。Nanos蛋白仅在胚胎的后极合成。大多数Nanos mRNA均匀分布在整个胚胎中，并在翻译上受到抑制。它的聚(A)尾巴被去掉(去烯基化)，RNA被缓慢降解。RNA的翻译抑制和降解都依赖于3‘UTR中的Smaug识别元件(SRE)，这些元件是调节蛋白Smaug的结合部位。依赖SRE的翻译抑制和去烯基化可以在果蝇早期胚胎的无细胞提取物中复制。已知去腺化是由CCR4-NOT复合体催化的。我们发现，翻译抑制涉及到在SRE上缓慢形成稳定的蛋白质复合体。该复合体含有7种蛋白质：Smaug、CUP、eIF4E、Me31B、Tral、PABPC和Belle。众所周知，杯赛会捆绑斯茂格。CUP还结合帽子结合的翻译起始因子eIF4E，并被认为竞争性地取代起始因子eIF4G。虽然这有助于抑制翻译，但抑制也可以在没有帽结构的情况下发挥作用，这表明还使用了第二种机制。我们已经纯化了抑制物复合体的七个成分，并能够在含有SRE的报告RNA上组装一个抑制物复合体，然后在随后的反应中抑制翻译。当使用某种细胞提取物来测定翻译时，Smaug和CUP足以在RNA上形成一个稳定的抑制物复合体；该抑制物复合体的额外成分可能是从翻译提取物中招募来的。本实验和其他实验确定Smaug-CUP-RNA复合体是被抑制的mRNP的核心。我们的建议旨在分析压制的机制。我们假设，一种双管齐下的机制正在运作，采用了如上所述的eIF4E结合，以及两个已知的翻译抑制因子Me31B和Tral的结合。我们将测试这两种蛋白质的复合体以核糖体无法获得的形式隔离RNA的想法。我们还将研究抑制物复合体的组件如何招募CCR4--而不是复合体。

Maternal mRNAs are RNAs that are provided from the mother to a developing oocyte. Such RNAs govern early development and are regulated by post-transcriptional mechanisms. One such RNA is the nanos mRNA, which encodes the determinant for posterior development of early Drosophila embryos. The Nanos protein is synthesized exclusively at the posterior pole of the embryo. Most of the nanos mRNA is homogeneously distributed throughout the embryo and is translationally repressed. Its poly(A) tail is removed (deadenylation), and the RNA is slowly degraded. Both translational repression and degradation of the RNA depend on Smaug Recognition Elements (SREs) in the 3‘ UTR, which are binding sites for the regulatory protein Smaug. SRE-dependent translational repression and deadenylation can be reproduced in a cell-free extract from early Drosophila embryos. Deadenylation is known to be catalyzed by the Ccr4-Not complex. We have found that translational repression involves the slow formation of a stable protein complex on the SREs. This complex contains seven proteins: Smaug, Cup, eIF4E, Me31B, Tral, PABPC and Belle. Cup is known to bind Smaug. Cup also binds the cap-binding translation initiation factor eIF4E and is thought to competitively displace the initiation factor eIF4G. While this contributes to the repression of translation, repression can also function in the absence of a cap structure, suggesting that a second mechanism is also used.We have purified the seven constituents of the repressor complex and are able to assemble a repressor complex on SRE-containing reporter RNAs that will then repress translation in a subsequent reaction. When a certain cell extract is used to assay translation, Smaug and Cup are sufficient for the formation of a stable repressor complex on the RNA; the additional components of the repressor complex are presumably recruited from the translation extract. This experiment and others identify the Smaug-Cup-RNA complex as the core of the repressed mRNP. Our proposal aims to analyze the mechanism of repression. We hypothesize that a two-pronged mechanism is operating, employing eIF4E binding as explained above and, in addition, the binding of Me31B and Tral, two known translation repressors. We will test the idea that a complex of these two proteins sequesters the RNA in a form that is inaccessible to ribosomes. We will also examine how components of the repressor complex recruit the CCR4-NOT complex.