Identification and characterization of novel components of a localized mRNP in Drosophila

黑腹果蝇（Drosophila melanogaster, Dm）的体轴形成需要母体mrna在卵母细胞中的不对称沉积。在定位转录本的最典型的例子是奥斯卡和双体mrna，其定位定义了前后极性。mRNA在细胞质中的定位取决于转录物的细胞核历史。定位mrna在细胞核转录过程中组装，在细胞核中，一组核心蛋白质与成熟mrna结合。这些蛋白为细胞质中更大的、动态的组件的形成提供了一个平台，这些组件调节mRNA的运输、沉默和本地化翻译。剪接mRNA的一个核心成分是外显子连接复合体（EJC），这是一种rna结合复合体，已知它可以招募其他蛋白质和蛋白质复合体，并且是oskar mRNA定位所必需的。EJC在后生动物中是保守的，因此构成了一个理想的模型系统来理解果蝇和高等真核生物的RNA定位机制，这一过程被认为是大脑发育中必不可少的。在糖尿病中，仅鉴定了EJC的少数结合伙伴，并且对含有EJC的mRNPs的确切分子组成，动力学和调控知之甚少。还不清楚哪些交互伙伴呈现EJC转录特异性。另一个存在于bicoid和oskar mRNP上的mRNP成分是Exuperantia蛋白（Exu），它是bicoid RNA定位所必需的。我们对Exu结构和功能的研究表明，Exu与未知结合伙伴的关联可能赋予转录特异性。迄今为止，还没有对纯化的原生oskar和bicoid mRNPs进行系统的生化研究以鉴定所有成分。然而，为了从功能和结构研究的重组蛋白中重建高阶组装，所涉及的蛋白质清单是至关重要的。我们的目标是使用定制的tap标记方法从转基因果蝇的卵母细胞中生化分离出定位的mRNPs。然后我们将使用质谱法来鉴定新的成分。我们将详细绘制体外mRNP组分之间的相互作用，并从重组蛋白中重建核心蛋白/RNA复合物。这将是进一步的结构-功能研究的起点。

Body axis formation in Drosophila melanogaster (Dm) requires the asymmetric deposition of maternal mRNAs in the oocyte. Among the best-characterized examples of localized transcripts are oskar and bicoid mRNAs, the localization of which defines antero-posterior polarity. mRNA localization in the cytoplasm depends on the nuclear history of the transcript. Localizing mRNPs assemble during transcription in the nucleus, where a core set of proteins binds to the maturing mRNAs. These proteins provide a platform for the formation of larger, dynamic assemblies in the cytoplasm that regulate mRNA transport, silencing and localized translation. One core component of spliced mRNPs is the exon junction complex (EJC), an RNA-binding complex that is known to recruit other proteins and protein complexes and that is required for localization of oskar mRNA. The EJC is conserved among metazoans and therefore constitutes an ideal model system to understand the mechanisms of RNA localization both in flies and in higher eukaryotes, where this process is thought to be essential in brain development. In Dm, only a few binding partners of the EJC have been identified and the exact molecular composition, dynamics and regulation of EJC-containing mRNPs is poorly understood. It is also unclear which interaction partners render the EJC transcript-specific. Another mRNP component present on both bicoid and oskar mRNPs is the protein Exuperantia (Exu), required for the localization of bicoid RNA. Our work on the structure and function of Exu suggests that association of Exu with an unknown binding partner might confer transcript-specificity. To date, no systematic biochemical studies on the purified native oskar and bicoid mRNPs have been performed to identify all components. However, to reconstitute higher order assemblies from recombinant proteins for functional and structural studies, an inventory of the proteins involved is critically important. Our aim is to biochemically isolate localized mRNPs from oocytes of transgenic flies using a customized TAP-tagging approach. We will then use mass spectrometry to identify novel components. We will map, in detail, the interactions between mRNP components in vitro and reconstitute core protein/RNA complexes from recombinant proteins. These will be a starting point for further structure-function studies.