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

果蝇体轴的形成需要母体RNA在卵母细胞中的不对称沉积。其中最典型的本地化转录本的例子是Oskar和Bigid mRNAs，它们的定位定义了前后两极。MRNA在细胞质中的定位取决于转录本的核历史。定位的mRNP在转录过程中在细胞核中组装，在那里一组核心蛋白质与成熟的mRNAs结合。这些蛋白质为在细胞质中形成更大的、动态的组装提供了一个平台，这些组装调控着mRNA的运输、沉默和局部翻译。剪接mRNPs的一个核心成分是外显子连接复合体(EJC)，这是一种已知招募其他蛋白质和蛋白质复合体的RNA结合复合体，是定位Oskar mRNA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式系统，以了解RNA在果蝇和高等真核生物中的定位机制，这一过程被认为是脑发育中必不可少的。在DM中，只确定了几个EJC的结合伙伴，并且对包含EJC的mRNPs的确切分子组成、动力学和调控知之甚少。目前也不清楚是哪些互动伙伴将EJC的记录具体化。另一个存在于双核糖体和奥斯卡核糖核酸上的mRNP成分是Exuperantia(Exu)蛋白，它是定位双核糖体RNA所必需的。我们对Exu结构和功能的研究表明，Exu与未知结合伙伴的结合可能赋予转录特异性。到目前为止，还没有对纯化的天然奥斯卡和双核rRNPs进行系统的生化研究来鉴定所有成分。然而，为了从重组蛋白质中重建更高阶的组装以进行功能和结构研究，所涉及的蛋白质的清单是至关重要的。我们的目标是使用定制的TAP标签方法从转基因果蝇的卵母细胞中生化分离定位的mRNPs。然后我们将使用质谱学来鉴定新的成分。我们将在体外详细绘制mRNP组件之间的相互作用图，并从重组蛋白中重建核心蛋白/RNA复合体。这些将是进一步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.