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: 10297652
• 负责人: Ahna Renee Skop
• 金额: $ 50.76万
• 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/10297652
• 关键词: Active Sites; Address; Affect; Anaphase; Aneuploidy; Architecture; Biogenesis; Biological Assay; Biological Response Modifier Therapy; Cell Proliferation; Cell division; Cells; Characteristics; Chemistry; Cilia; Clustered Regularly Interspaced Short Palindromic Repeats; Coculture Techniques; Color; Complement; Complex; Coupled; Cytokinesis; Cytoplasm; Cytoplasmic Granules; Data; Disease; Double-Stranded RNA; Electrons; Embryo; Endoderm Cell; Excision; Exhibits; Genomics; Goals; Hela Cells; Human; Instruction; 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; Plant Roots; Play; Proteins; Publishing; RNA; RNA Transport; Regulation; Reporting; Research; Resolution; Reverse Transcriptase Polymerase Chain Reaction; Ribonucleoproteins; Role; Signal Transduction; Site; Structure; Syndrome; System; Tissues; Transcript; Translating; Translations; Work; base; biophysical properties; cell type; common treatment; daughter cell; extracellular; extracellular vesicles; genetic manipulation; human disease; innovation; insight; intercellular communication; novel; pluripotency; scaffold; spatiotemporal; stem cells; therapeutic target; transcription factor; transcriptome; transcriptome sequencing; tumorigenesis

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，即脱落，依赖于一种称为中体的瞬时结构，它位于细胞内部 新形成的子细胞之间的细胞间桥梁。它由重叠的纺锤体中区组成 微管涂有称为中体基质的电子致密材料。长期概念化为 胞质分裂后的结构残余物会发生降解，新出现的数据表明中间体发挥着作用 有丝分裂后在建立细胞命运、增殖状态、组织极性、纤毛形成、神经元方面的指导作用 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，因为它首先由 Michael Mullins 和 Dick McIntosh 在 20 世纪 70 年代积极追求，然后由 20世纪80年代的栗山凉子。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 因为它是 RNA 储存位点，并且是一种新型主动翻译 RNP 颗粒，具有独特的复杂生命 循环由无膜相和膜结合相组成，我们称之为 MB 颗粒（对于 中体颗粒）。在活细胞和固定细胞中采用定量、超分辨率方法，并结合 通过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 中体 RNP 颗粒周围的隔室，以及有丝分裂后中体残余物（或 MBR）， 和内化 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，但是 推测是在有丝分裂后起作用。富含中体的转录本在脱落前立即启动翻译， 我们所展示的这一步骤发生在 G1 早期，就在核膜完全重新组装之后。治疗方法 通常用于确定 RNP 相凝结物，揭示了中间体基质的行为就像一种新颖的 类型的 RNP 颗粒，并且关键的胞质分裂驱动蛋白马达 Kif23/MKLP1 可能作为一种新型 RNP 脚手架。 MB 颗粒是一类非常新颖的 RNP 颗粒，因为它具有翻译活性，具有膜- 少相和膜结合相，并在细胞命运和增殖决策中有丝分裂后发挥作用。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被释放。我们的 拟议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新型细胞间质表现 沟通。