The roles of midbody associated mRNAs in regulating cell proliferation and differentiation

中体相关mRNA在调节细胞增殖和分化中的作用

• 批准号: 10725063
• 负责人: Rytis Prekeris
• 金额: $ 7.32万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-20 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10725063
• 关键词: 3' Untranslated Regions; Apical; Binding Proteins; Biological Assay; CENP-E protein; CRISPR screen; Cell Cycle Stage; Cell Differentiation process; Cell Proliferation; Cell division; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Cues; Cytokinesis; Data; Diffusion; Electron Microscopy; Elements; Excision; Extracellular Space; Foundations; Goals; Image; Immunofluorescence Immunologic; Interphase; Interphase Cell; Knowledge; Laboratories; Location; Maps; Mediating; Messenger RNA; Microscopy; Microtubules; Mitosis; Mitotic; Molecular; Mothers; Mutagenesis; Neurites; Organelles; Play; Process; Proliferating; Proteins; Proteomics; Protocols documentation; RNA-Binding Proteins; Resolution; Role; Side; Signal Pathway; Signal Transduction; Stimulation of Cell Proliferation; Structure; Testing; Thinness; Translating; Translations; Untranslated Regions; cancer cell; daughter cell; light microscopy; member; novel; physical separation; postmitotic; recruit; stem cells; transcriptome sequencing; uptake

Project Summary During mitosis, the mother cell divides by the formation of a cleavage furrow, leaving two daughter cells connected by a thin intercellular bridge. The resolution of this bridge, abscission, leads to the separation of the two daughter cells. During ingression of the cleavage furrow, the central spindle microtubules are compacted to form a structure known as the midbody (MB). It is now well established that MB regulates cytokinesis by recruiting abscission-mediating proteins, such as ESCRT complex, as well as several regulators of abscission checkpoint. Until recently, the MB was thought to be discarded after division by releasing it into extracellular space. However, recently it was shown that MBs accumulate in stem and cancer cells after mitosis has been completed (called MBsomes) and it has been proposed that MBsomes function as novel signalling platforms that regulate cell differentiation and proliferation. Recently we developed a protocol for purification of post- mitotic MBs and completed their proteomic and RNAseq analyses that led to identification of several mRNAs and mRNA-binding proteins that accumulate at the MB. Importantly, MB-enriched mRNAs encode several ESCRT complex subunits, as well as proteins that stimulate cell proliferation. Furthermore, we show that these MB-enriched mRNAs can be transferred to the neighboring cells via post-mitotic MB internalization. Based on all of these findings, we hypothesize that targeting of selected mRNAs to the MB during cytokinesis play a key role in regulating cell abcission and post-mitotic MBsome signaling. Here we propose three specific aims to test this hypothesis. First, we will map and characterize the domain(s) within 3'-UTR that are needed for mRNA targeting during cytokinesis. We will then use candidate approach, as well as proteomic and CRISPR screens, to identify RNA-binding proteins that interact with these 3”-UTR domains and regulate mRNA targeting and localized translation at the MB. Second, we will test the possibility that MB accumulation/translation of ESCRT mRNAs mediates ESCRP complex targeting to the MB. Third, we will test whether MBsome-dependent transfer of specific mRNAs, such as mRNA encoding proliferation regulator CENP-E contribute to MBsome-induced cell proliferation.

项目摘要 在有丝分裂过程中，母细胞通过形成卵裂沟而分裂，留下两个子细胞 由一个细的细胞间桥连接。这座桥的解决方案，分离，导致分离的 两个子细胞在卵裂沟的侵入过程中，纺锤体微管被压缩 以形成称为中间体（MB）的结构。现在已经确定MB通过以下方式调节胞质分裂： 募集脱落介导蛋白，如ESCRT复合物，以及几种脱落调节因子 检查站直到最近，MB被认为是在分裂后通过释放到细胞外而被丢弃的。 空间然而，最近的研究表明，在有丝分裂结束后，MB在干细胞和癌细胞中积累。 已经完成了（称为MBsomes），并且已经提出MBsomes作为新的信号平台发挥作用 调节细胞分化和增殖。最近，我们开发了一种用于纯化后- 有丝分裂MB，并完成了其蛋白质组学和RNAseq分析，从而鉴定了几种mRNA 以及在MB处积累的mRNA结合蛋白。重要的是，MB富集的mRNA编码几种 ESCRT复合物亚基，以及刺激细胞增殖的蛋白质。此外，我们表明，这些 MB富集的mRNA可以通过有丝分裂后MB内化转移到邻近细胞。基于 所有这些发现，我们假设在胞质分裂期间将选定的mRNA靶向MB发挥作用 在调节细胞脱落和有丝分裂后MBsome信号传导中起关键作用。在这里，我们提出三个 具体目的是检验这一假设。首先，我们将定位和表征3 '-UTR内的结构域， 在胞质分裂期间需要mRNA靶向。然后，我们将使用候选方法，以及蛋白质组学和 CRISPR筛选，以鉴定与这些3”-UTR结构域相互作用并调节 mRNA靶向和MB处的定位翻译。第二，我们将测试MB ESCRT mRNA的积累/翻译介导ESCRP复合物靶向MB。第三，我们将测试 是否MBsome-dependent转移的特定mRNA，如mRNA编码的增殖调节 CENP-E有助于MBsome诱导的细胞增殖。