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.

概括 细胞分裂的最后一步，脱落，依赖于一种称为中体的瞬态结构，该结构内部的住宅 新形成的子细胞之间的细胞间桥。它由重叠的纺锤体中区组成 涂有电子密集材料的微管，称为中体基质。长期概念化为 结构性残留物可能会在细胞因子后降解，新出现的数据表明中等体系 有启发性的有丝分裂后作用在建立细胞命运，增殖状态，组织极性，纤毛形成，神经元中 功能和肿瘤发生。但是，关于电子密度的功能意义知之甚少 材料，因为它是迈克尔·穆林斯（Michael Mullins）和迪克·麦金托什（Dick McIntosh）在1970年代首次积极追求的，然后是 Ryoko Kuriyama在1980年代。我的实验室发现了这种电子致密材料的令人惊讶的新功能 这是一个RNA存储的位置，是一种活跃地翻译RNP颗粒的新颖的，具有独特的复杂寿命 我们称为MB颗粒的循环完成了无膜和膜结合的阶段（用于 中体颗粒）。在活细胞和固定细胞中采用定量的超分辨率方法，并与 基因组学和遗传操纵以解决我们的问题，我们发现翻译发生在一个 围绕中体RNP颗粒以及有丝分裂后的中体残留物（或MBR）的隔室， 和内部MBR（或MBSOMES）。分离的哺乳动物中间体的RNA-seq数据揭示了一种酶 在细胞分裂中未描述功能的致癌和干细胞转录因子的 大概是在有效的。富含中体的成绩单在脱落前立即启动翻译， 在核包膜完全重新组合后，我们在G1早期显示的步骤。治疗 通常用于确定RNP相冷凝水的通常用于表明中体矩阵作为一种新颖 RNP颗粒的类型，以及关键的细胞因子运动蛋白运动蛋白MOTOT，KIF23/MKLP1，可以用作新的RNP 脚手架。 MB颗粒是一种非常新颖的RNP颗粒类别，因为它具有翻译活性，具有膜 - 较少的和膜结合的相位，并在细胞命运和增殖的决策中发挥作用。这里， 我们将集中精力确定MBR的细胞类型特定成分，即时空调节 MBSOME结构和功能，以及如果MB颗粒RNA在吞噬它们的细胞中被释放。我们的 拟议的研究将发现对组成和不同的MBSOME结构和功能的独特见解， 提供有关这种独特的RNP凝结物和细胞器的表现的洞察力 沟通。

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.