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 细胞分裂的最后一步是分裂，它依赖于一种叫做中间体的临时结构， 新形成的子细胞之间的细胞间桥梁。它由重叠的纺锤体中间区组成 微管被称为中间体基质的电子致密物质包裹。长期概念化为 一个结构上的残余物受到降解后，细胞质分裂，新兴的数据表明， 在建立细胞命运、增殖状态、组织极性、纤毛形成、神经元 功能和肿瘤发生。然而，人们对电子密度的功能意义知之甚少。 材料，因为它是第一次积极追求迈克尔马林斯和迪克麦金托什在20世纪70年代，然后由 栗山凉子在1980年代。我的实验室发现了这种电子密度高的材料的一个令人惊讶的新功能 因为它是RNA储存位点，是一种具有独特复杂生命的新型主动翻译RNP颗粒 一个由无膜和膜结合相组成的循环，我们称之为MB颗粒（用于 中间体-颗粒）。在活细胞和固定细胞中采用定量、超分辨率方法， 基因组学和基因操作来解决我们的问题，我们发现翻译发生在一个 在围绕中间体RNP颗粒的隔室中，以及在有丝分裂后的中间体残余物（或MBR）中， 和内化的MBR（或MBsome）。分离的哺乳动物中间体的RNA-seq数据揭示了富集 致癌和干细胞转录因子在细胞分裂中没有描述的功能，但 可能是在有丝分裂后起作用。中间体富集的转录物在翻译前立即启动翻译， 我们已经展示的一个步骤发生在G1早期，就在核膜完全重组之后。治疗 通常用于确定RNP相冷凝物显示，中间体基质表现为一种新的 Kif 23/MKLP 1可能是一种新的RNP， 脚手架MB-颗粒是一类非常新颖的RNP颗粒，因为它具有免疫活性，具有膜- 较少和膜结合期，并在细胞命运和增殖决定中起有丝分裂后的作用。在这里， 我们将集中精力确定MBR的细胞类型特异性成分，时空调节， MBsome的结构和功能，以及MB颗粒RNA是否在吞噬它们的细胞中释放。我们 提出的研究将揭示对保守和不同的MBsome结构和功能的独特见解， 深入了解这种独特的RNP凝聚物和细胞器如何作为细胞间的新形式 通信

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.