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: 10436326
• 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/10436326
• 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细胞分裂和长期记忆形成，如 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 与核蛋白和细胞质蛋白的相互作用是 mRNA 有效转运至亚细胞所必需的。 细胞区域。最近，参与加工体（P-体）形成的因素也已被 连接到这个过程。生化和遗传数据揭示了与 mRNA 在其生命周期中，但破译这些动态和时间的时空要求 短暂的相互作用只能通过体内直接观察来实现。 过去十年，我们在检测单个 mRNP 复合物方面取得了重大进展 在体内，使用黑腹果蝇卵室和分子信标技术。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。该实验设置解决了关键动态事件背后的细节，包括 运输过程中 mRNA 的翻译控制和蛋白质 mRNA 的时空测定 关联和解离。 我们新颖的中心假设是，P 体的形成是由初始成核事件控制的 关键的核心支架因素，然后是招募表现出不同的外壳客户成员 生物物理特征，这反过来又协调了母体 mRNA 的亚细胞命运 在多 mRNA 物种复合物中运输。为此，我们启动了研究，以确定 P- 体组装的发生以及 P 体在 D 中转录本运输过程中所扮演的角色。 黑腹果蝇卵室。该提案的目标是描述多个 P 体如何 成员和局部 mRNA 转录本在空间和时间上进行组织，从而给出了很多- 对相互关联和相互依存之间的机制联系有必要的理解水平 mRNA 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。通过将分子信标技术和单分子RNA FISH探针与先进的技术相结合 成像方法，我们将实现多个母体 mRNA 和 P-的同时可视化 首次以高分辨率显示身体蛋白质。使用互补的生化和生物物理测定， 我们将进一步对P-body成分进行整理和分类，以揭示它们参与RNA依赖的过程 流程。这些研究将使我们能够探索基因背后的新分子机制 涉及 P 体的表达，因此将具有细胞生物学之外的深远影响 研究领域包括病毒复制、肿瘤形成、衰老和神经退行性疾病。