Characterizing P-bodies assembly and coordination of mRNA fate during Drosophila melanogaster oogenesis

1.摘要 基因表达调控的一个重要机制是信使的亚细胞定位。 RNA(MRNAs)。不正确的定位会扰乱不对称的细胞分裂，即长期记忆的形成，因为 以及在早期发育过程中后生动物体型的建立。高度协调 与核蛋白和细胞质蛋白的相互作用是将mRNAs有效地转运到 细胞区域。最近，参与处理小体(P小体)形成的因素也被 已连接到此进程。生化和基于遗传的数据揭示了与 在其生命周期中，但破译了这些动态和 短暂的相互作用只能通过在体内直接观察才能实现。 在过去的十年中，我们在检测单个mRNP复合体方面取得了重大进展 在活体内，使用黑腹果蝇卵室和分子信标技术。合作的能力- 实时可视化和跟踪亚细胞空间内的mRNPs一直是研究的无价资产 RNA过程。此实验设置解决了关键动态事件背后的细节，包括 MRNAs转运过程中的翻译调控及蛋白质-mRNA的时空测定 关联和解除关联。 我们新的中心假设是，P体的形成是由初始成核事件通过 关键的核心-脚手架因素，其次是招募表现不同的壳客户成员 生物物理特性，这又协调了母体mrna的亚细胞命运。 在多个信使核糖核酸物种复合体中运输。为此，我们启动了一些研究，以确定P- 小体的组装以及P小体在转录本运输中的作用(S)。 黑腹蛇的卵室。该提案目的是描述多个P-Body如何 成员和本地化的信使核糖核酸转录本在空间和时间上被组织起来，从而提供了许多- 对相互联系和相互依赖之间的机械联系有必要的理解程度 真核生物中重要的信使核糖核酸的运输、储存、翻译抑制和定位过程 生活。通过将分子信标技术和单分子RNA FISH探针与先进的 成像方法，我们将实现多个母体mRNAs和P- 首次以高分辨率显示人体蛋白质。使用互补的生化和生物物理分析， 我们将进一步整理和分类P-Body成分，以揭示它们参与RNA依赖 流程。这些研究将使我们能够探索基因背后的一种新的分子机制 因此，涉及P-小体的表达将具有超越细胞生物学的深远影响 研究，包括病毒复制、肿瘤形成、衰老和神经退行性疾病。

1. Abstract An important mechanism of gene expression regulation is the subcellular localization of messenger RNAs (mRNAs). Incorrect localization disrupts asymmetric cell division, long-term memory formation, as well as the establishment of metazoan body patterning during early development. Highly coordinated interactions with nuclear and cytoplasmic proteins are required for efficient transport of mRNAs to sub- cellular regions. More recently, factors involved in Processing body (P-body) formation have also been connected to this process. Biochemical and genetic-based data have revealed key factors associated with an mRNA during its life cycle, but deciphering the spatial-temporal requirements of these dynamic and ephemeral interactions can only be achieved by direct observation in vivo. We have made significant advances over the last decade in detecting individual mRNP complexes in vivo, using D. melanogaster egg chambers and the molecular beacon technology. The ability to co- visualize and track mRNPs within subcellular space in real time has been an invaluable asset for studying RNA processes. This experimental setup has resolved details behind key dynamic events, including translational control of mRNAs during transport and spatiotemporal determination of protein-mRNA association and disassociation. Our novel central hypothesis is that formation of P bodies is governed by initial nucleation events via key core-scaffold factors, followed by the recruitment of shell-client members that exhibit different biophysical characteristics, This, in turn, coordinates the subcellular fate of maternal mRNAs as they are transported in multi-mRNA species complexes. To this end, we initiated studies that will determine how P- body assembly takes place and the role(s) played by P-bodies during transcript transport in D. melanogaster egg chambers. The objective of this proposal is to characterize how multiple P-body members and localized mRNA transcripts are spatially and temporally organized, thus giving a much- needed level of understanding of the mechanistic links between interconnected and interdependent processes of mRNA transport, storage, translational repression and localization important in all eukaryotic life. By integrating the molecular beacon technology and single-molecule RNA FISH probes with advanced imaging approaches, we will achieve the simultaneous visualization of multiple maternal mRNAs and P- body proteins at high resolution for the first time. Using complementary biochemical and biophysical assays, we will further sort out and classify P-body components to reveal their involvement in RNA-dependent processes. These studies will enable us to explore a novel molecular mechanism underlying gene expression that involving P-bodies, and thus, will have far-reaching implications beyond cell biology research, including viral replication, tumor formation, aging and neurodegenerative diseases.