Functional analysis of mammalian midbody RNA in post-mitotic signaling functions

哺乳动物中间体 RNA 在有丝分裂后信号传导功能中的功能分析

• 批准号: 10684068
• 负责人: Ahna Renee Skop
• 金额: $ 41.52万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-16 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10684068
• 关键词: Active Sites; Address; Affect; Anaphase; Aneuploidy; Architecture; Binding; Biogenesis; Biological Assay; Biological Response Modifier Therapy; Cell division; Cells; Characteristics; Chemistry; Cilia; Clustered Regularly Interspaced Short Palindromic Repeats; Color; Complement; Complex; Coupled; Cytokinesis; Cytoplasm; Cytoplasmic Granules; Data; Disease; Double-Stranded RNA; Electrons; Embryo; Excision; Exhibits; Genomics; Goals; Hela Cells; Human; Kinesin; Knock-out; LOX gene; Life Cycle Stages; Malignant Neoplasms; Mediating; Membrane; Messenger RNA; Microcephaly; Microtubules; Mitosis; Mitotic; Modeling; Molecular; Motor; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Nuclear Envelope; Oncogenic; Organelles; Phase; Physical condensation; Play; Proliferating; Proteins; Publishing; RNA; RNA Transport; Regulation; Reporting; Research; Reverse Transcriptase Polymerase Chain Reaction; Ribonucleoproteins; Role; Signal Transduction; Site; Structure; Syndrome; System; Tissues; Transcript; Translating; Translation Initiation; Translations; Work; biophysical properties; cell fixing; cell type; common treatment; daughter cell; delivery vehicle; extracellular; extracellular vesicles; genetic manipulation; human disease; innovation; insight; intercellular communication; novel; pluripotency; postmitotic; scaffold; spatiotemporal; stem cells; therapeutic target; transcription factor; transcriptome sequencing; transcriptomic profiling; tumorigenesis; ultra high resolution

SUMMARY The last step in cell division, abscission, relies on a transient structure called the midbody, which resides inside the intercellular bridge between newly forming daughter cells. It consists of overlapping spindle midzone microtubules which are coated with electron dense material called the midbody matrix. Long conceptualized as a structural remnant subject to degradation following cytokinesis, emerging data suggest that midbodies play instructive post-mitotic roles in establishing cell fate, proliferation state, tissue polarity, cilia formation, neuron function, and oncogenesis. However, very little is known about the functional significance of the electron dense material, since it was first actively pursued by Michael Mullins and Dick McIntosh in the 1970s, and then by Ryoko Kuriyama in the 1980s. My lab has uncovered a surprising novel function for this electron dense material in that it is a site of RNA storage and is a novel actively translating RNP granule with a uniquely complex life cycle comprised of both membrane-less and membrane-bound phases, that we are calling the MB-granule (for Midbody-granule). Employing quantitative, super-resolution approaches in live and fixed cells, coupled with genomics and genetic manipulations to address our questions, we discovered that translation occurs in a compartment surrounding the midbody RNP granule, as well as in post-mitotic midbody remnants (or MBRs), and internalized MBRs (or MBsomes). RNA-seq data of isolated mammalian midbodies revealed an enrichment of both oncogenic and stem cell transcription factors that have no described function in cell division, but presumably act post-mitotically. Midbody-enriched transcripts initiate translation immediately before abscission, a step that we have shown occurs in early G1, just after the nuclear envelope is fully reassembled. Treatments commonly used to determine RNP phase condensates revealed that the midbody matrix behaves as a novel type of RNP granule, and that the critical cytokinesis kinesin motor, Kif23/MKLP1, may serve as a novel RNP scaffold. The MB-granule is a very novel class of RNP granule in that it’s translationally active, has membrane- less and membrane-bound phases, and functions post-mitotically in cell fate and proliferative decisions. Here, we will focus our efforts to determine the cell-type specific components of MBRs, the spatiotemporal regulation of MBsome structure, and function, and if MB-granule RNAs are being liberated in cells that engulf them. Our proposed studies will uncover unique insights into conserved and divergent MBsome structure and function, offering insight into how this unique RNP condensate and organelle behaves as novel form of intercellular communication.

总结

项目成果

A protocol for isolating and imaging large extracellular vesicles or midbody remnants from mammalian cell culture.

• DOI: 10.1016/j.xpro.2023.102562
• 发表时间: 2023-12-15
• 期刊: STAR PROTOCOLS
• 作者: Park, Sungjin;Patel, Smit A.;Torr, Elizabeth E.;Dureke, Ashley-Grace N.;Mcintyre, Alina M.;Skop, Ahna R.
• 通讯作者: Skop, Ahna R.