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