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

果蝇卵子发生过程中 P 体组装和 mRNA 命运协调的特征

• 批准号: 10212422
• 负责人: Diana P. Bratu
• 金额: $ 39万
• 依托单位: HUNTER COLLEGE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-07 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10212422
• 关键词: Address; Aging; Biochemical; Biochemical Genetics; Biological Assay; Biology; Biophysics; Body Composition; Body Patterning; Cell division; Cell physiology; Cellular biology; Client; Code; Complement; Complex; Confocal Microscopy; Coupled; Cytoplasmic Granules; Cytoplasmic Protein; Data; Dendrites; Development; Drosophila melanogaster; Electrons; Event; Exhibits; Fluorescent in Situ Hybridization; Functional disorder; Gene Expression; Gene Expression Regulation; Genes; Genetic Databases; Genetic Transcription; Goals; Imaging Techniques; Individual; Life; Life Cycle Stages; Link; Liquid substance; Maintenance; Maternal Messenger RNA; Messenger RNA; Microscopy; Modeling; Molecular; Morphology; Nature; Neurodegenerative Disorders; Neurons; Nuclear Proteins; Onset of illness; Oocytes; Oogenesis; Pathology; Peptide Initiation Factors; Phase; Play; Post-Transcriptional Regulation; Process; Property; Proteins; RNA; RNA Transport; Regulation; Regulon; Research; Resolution; Role; Shapes; Structure; Subcellular Spaces; Technology; Time; Transcript; Translational Repression; Translations; Virus Replication; Visualization; Work; axon guidance; biophysical properties; cell type; egg; imaging approach; in vivo; insight; long term memory; member; neuronal cell body; novel; novel therapeutics; protein complex; recruit; scaffold; single molecule; spatial relationship; spatiotemporal; tumor; tumorigenesis

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.

1。摘要 基因表达调控的重要机制是信使的亚细胞定位 RNA（mRNA）。不正确的定位破坏了不对称的细胞分裂，长期记忆形成，如 以及在早期发育过程中建立后生动物体图案的建立。高度协调 与核和细胞质蛋白相互作用是有效地将mRNA传递到亚 细胞区域。最近，涉及加工体（P-Body）形成的因素也已经存在 连接到此过程。生化和基于遗传的数据揭示了与 在其生命周期中的mRNA，但解读了这些动态和 短暂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步 在体内，使用D. melanogaster鸡蛋室和分子信标技术。共同的能力 实时可视化和跟踪亚细胞空间内的mRNP已成为研究的宝贵资产 RNA过程。这种实验设置已解决关键动态事件背后的细节，包括 在运输和时空测定蛋白-MRNA期间mRNA的翻译控制 关联和分离。 我们新颖的中心假设是，P体的形成受初始成核事件的控制 关键的核心损伤因素，然后招募表现出不同的壳牌成员 生物物理特征，这反过 在多MRNA物种复合物中运输。为此，我们启动了研究，这些研究将决定p- 身体组装发生，并且在D。 Melanogaster鸡蛋室。该建议的目的是表征多个P体的方式 成员和局部mRNA转录本在空间和时间上都是组织的，因此给出了很多 所需的对互连与相互依赖之间机械联系的理解水平 mRNA传输，存储，翻译抑制和本地化的过程在所有真核过程中都很重要 生活。通过将分子信标技术和单分子RNA鱼探针与先进 成像方法，我们将实现多种母体mRNA和p-的同时可视化 首次以高分辨率的身体蛋白质。使用互补的生化和生物物理测定法， 我们将进一步整理并分类P体分，以揭示其参与RNA依赖性 过程。这些研究将使我们能够探索一种新的分子机制。 涉及p-体以及因此将具有细胞生物学以外的深远影响的表达 研究，包括病毒复制，肿瘤形成，衰老和神经退行性疾病。