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（mRNA）。不正确的定位会破坏不对称的细胞分裂，长期记忆的形成， 以及在早期发育过程中后生动物身体模式的建立。高度协调 与细胞核和细胞质蛋白的相互作用是mRNA有效转运到亚细胞所必需的。 细胞区域。最近，涉及加工体（P体）形成的因素也已被研究。 与这个过程有关。基于生物化学和遗传学的数据揭示了 mRNA在其生命周期中，但破译这些动态的时空要求， 短暂的相互作用只能通过体内直接观察来实现。 在过去的十年中，我们在检测单个mRNP复合物方面取得了重大进展。 在体内，使用D.黑腹虫卵室和分子信标技术。合作的能力- 在亚细胞空间内真实的时间内可视化和跟踪mRNP已经成为研究的宝贵资产 RNA处理。这个实验性的设置解决了关键动态事件背后的细节，包括 转运过程中mRNA的翻译控制和蛋白质-mRNA的时空测定 关联和分离。 我们新的中心假设是，P体的形成是由初始成核事件通过 关键的核心支架因素，其次是招募壳客户端成员，表现出不同的 生物物理特性，这反过来又协调了母体mRNA的亚细胞命运，因为它们是 在多mRNA物种复合物中转运。为此，我们启动了研究，以确定如何P- 体的组装以及P体在D. 黑腹产卵室本提案的目的是描述多个P体 成员和定位的mRNA转录本在空间和时间上是有组织的，因此给出了一个非常- 对相互联系和相互依赖之间的机械联系的必要理解水平 mRNA的运输、储存、翻译抑制和定位过程在所有真核生物中都很重要。 生活通过整合分子信标技术和单分子RNA FISH探针， 成像方法，我们将实现多个母体mRNA和P- 第一次以高分辨率显示人体蛋白质。使用互补的生物化学和生物物理测定， 我们将进一步整理和分类P体成分，以揭示它们参与RNA依赖的 流程.这些研究将使我们能够探索一种新的分子机制， 涉及P体的表达，因此，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.