mRNP assembly and remodeling for transport and translational control in Drosophila

用于果蝇运输和翻译控制的 mRNP 组装和重塑

• 批准号: 283135182
• 负责人: Dr. Anne Ephrussi, Ph.D.
• 金额: --
• 依托单位: Europäisches Laboratorium für Molekularbiologie (EMBL)
• 依托单位国家: 德国
• 项目类别: Research Units
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/283135182?language=en
• 关键词: mRNP; assembly; remodeling; transport; translational

Cytoplasmic mRNA localization is a powerful and conserved mechanism for achieving localized protein expression in eukaryotic cells; it is prevalent in Drosophila, whose oocyte-localized transcripts encoding embryonic axis determinants have become models for the study of mRNA transport, local translation and anchoring. With its highly developed tools for experimental analysis, Drosophila is an ideal model system for biochemical, genetic and cell biological investigation of mRNA localization mechanisms as they occur in vivo. A paradigm is Drosophila oskar mRNA, whose localization is a multi-step process that includes: (1) nuclear splicing-dependent generation of a bipartite localization signal within the oskar mRNA coding region and (2) concomitant, splicing dependent deposition of the Exon Junction Complex (EJC) on the mRNA; (3) cytoplasmic recruitment of proteins required for (4) microtubule motor-dependent transport of oskar messenger ribonucleoproteins (mRNPs) from the nurse cells into the oocyte by the microtubule motor, dynein and to the posterior pole by kinesin-1; (5) translational repression of the mRNA in transport and (6) localized translational activation of oskar mRNA at the oocyte posterior, which is required for the (7) Oskar protein dependent mRNA anchoring at the posterior pole. While (mainly) genetics has led to the identification of a few of the proteins involved in this complex regulation, neither is the full complement of proteins known, nor are how and when they (inter)act in the process understood. As a first aim of our project, we plan to address the broader role of the Drosophila EJC in mRNA localization in the fly and to understand cellular events and molecular features that determine its stable and selective binding to specific sites on mRNAs. The second aim of the project is to develop and apply a method for transcript- and translational status-specific purification of intracellular transport mRNPs in Drosophila. As a proof of principle, we will use oskar mRNA, then extend to other Drosophila localized mRNAs towards a systemic view of localization mRNP composition and its functional analysis in vivo in the fly.

细胞质mRNA定位是一种在真核细胞中实现局部蛋白质表达的强大而保守的机制。它在果蝇中很普遍，果蝇的卵母细胞定位的转录本编码胚胎轴决定因素已成为研究mRNA转运，局部翻译和锚定的模型。果蝇凭借其高度开发的实验分析工具，是一种理想的模型系统，用于在体内发生的mRNA定位机制的生化，遗传和细胞生物学研究。范式是果蝇Oskar mRNA，其定位是一个多步骤过程，其中包括：（1）Oskar mRNA编码区域内的核剪接依赖性产生的核剪接依赖性产生，以及（2）（2）伴随的，（2）偶联的，依赖于外显子交界复合物（EJC）在mRNA上的拼接依赖性沉积； （3）（4）（4）微管运动依赖性转运Oskar Messenger核糖核蛋白（MRNPS）从护士细胞通过微管运动，Dynein和kinesin-pole的微管向卵母细胞的细胞质募集； （5）转运中mRNA的翻译抑制和（6）Oskar mRNA在卵母细胞后部的局部翻译激活，这是（7）Oskar蛋白依赖的mRNA所必需的，锚定在后极。虽然（主要）遗传学导致鉴定了这种复杂调节所涉及的一些蛋白质，但已知蛋白质的完整补体也不是（Inter）在该过程中如何和何时起作用。作为我们项目的第一个目的，我们计划解决果蝇EJC在果蝇中的mRNA定位中的更广泛作用，并了解确定其稳定和选择性结合在mRNA上的特定位点的细胞事件和分子特征。该项目的第二个目的是开发和应用一种果蝇中细胞内转运mRNP的转录和翻译状态特异性纯化的方法。作为原则的证明，我们将使用Oskar mRNA，然后延伸到其他果蝇局部的mRNA，朝着局部MRNP组成的系统性视图及其在Vivo中的功能分析。