Formation of the repressed nanos mRNP in the early Drosophila embryo

母体mRNA是从母体提供给发育中的卵母细胞的RNA。这些RNA控制早期发育，并受转录后机制调控。一种这样的RNA是nanos mRNA，它编码早期果蝇胚胎后部发育的决定因素。Nanos蛋白只在胚胎的后极合成。大多数nanos mRNA均匀地分布在整个胚胎中，并受到抑制。它的poly（A）尾被去除（去腺苷酸化），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），其是调节蛋白Smaug的结合位点。SRE依赖的翻译抑制和去腺苷化可以在早期果蝇胚胎的无细胞提取物中复制。已知脱腺苷酸化由Ccr 4-Not复合物催化。我们已经发现，翻译抑制涉及一个稳定的蛋白质复合物的SRE上的缓慢形成。该复合物包含7种蛋白质：Smaug、Cup、eIF 4 E、Me 31 B、Tral、PABPC和贝儿。圣杯可以束缚史矛革Cup还结合帽结合翻译起始因子eIF 4 E，并被认为竞争性取代起始因子eIF 4G。虽然这有助于抑制翻译，抑制也可以在没有帽结构的情况下发挥作用，这表明第二种机制也是used.We已经纯化的阻遏物复合物的七种成分，并能够组装一个阻遏物复合物的SRE-含有报告RNA，然后将在随后的反应中抑制翻译。当某种细胞提取物用于检测翻译时，Smaug和Cup足以在RNA上形成稳定的阻遏物复合物;阻遏物复合物的其他组分可能是从翻译提取物中招募的。该实验和其他实验将Smaug-Cup-RNA复合物鉴定为受抑制的mRNP的核心。我们的建议旨在分析压抑的机制。我们假设一个双管齐下的机制正在运作，采用如上所述的eIF 4 E结合，此外，Me 31 B和Tral，两个已知的翻译阻遏物的结合。我们将测试这两种蛋白质的复合物以核糖体无法接近的形式螯合RNA的想法。我们还将研究阻遏物复合物的组成部分如何募集CCR 4-NOT复合物。

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.