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: 10624620
• 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/10624620
• 关键词: Binding Proteins; CENP-E protein; CRISPR screen; Cell Differentiation process; Cell Proliferation; Cells; Complex; Cytokinesis; Excision; Extracellular Space; Goals; Maps; Mediating; Messenger RNA; Microtubules; Mitosis; Mitotic; Molecular; Mothers; Organelles; Play; Proteins; Proteomics; Protocols documentation; RNA-Binding Proteins; Resolution; Role; Signal Pathway; Signal Transduction; Structure; System; Testing; Thinness; Translations; Untranslated Regions; base; cancer cell; daughter cell; novel; recruit; stem cells; transcriptome sequencing

Project Summary This Project Summary/Abstract was originally submitted with R01 GM143774 and is included here unchanged to satisfy submission system requirements. 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 该项目摘要/摘要最初是用R01 GM143774提交的，此处包括 满足提交系统的要求。 在有丝分裂期间，母细胞除以裂解沟的形成，留下两个子细胞 由薄细胞间桥连接。该桥的分辨率，脱落，导致分离 两个子细胞。在裂解沟的插入过程中，中央纺锤微管被压实 形成一个称为中体（MB）的结构。现在已经确定MB可以通过 募集脱脱脱发的蛋白质（例如ESCRT复合物）以及脱落的几个调节剂 检查点。直到最近，人们认为MB通过将其释放成细胞外丢弃。 空间。然而，最近显示，有丝分裂后的茎和癌细胞中积累的MB一直是 已完成（称为MBSOMES），已经提出MBSOMES作为新的信号平台 调节细胞分化和增殖。最近，我们开发了一种净化后的协议 有丝分裂MB并完成其蛋白质组学和RNASEQ分析，这些分析导致了几种mRNA 和积聚在MB的mRNA结合蛋白。重要的是，富含MB的mRNA编码几个 ESCRT复合体亚基以及刺激细胞增殖的蛋白质。此外，我们证明了这些 富含MB的mRNA可以通过有丝分裂后MB内在化转移到相邻细胞中。基于 所有这些发现，我们假设在细胞因子游戏中靶向选定的mRNA对MB的靶向 控制细胞脱落和有丝分裂后MBSOME信号传导的关键作用。在这里我们提出三个 具体旨在检验这一假设的目的。首先，我们将映射和表征3'-UTR中的域 在细胞因子过程中需要mRNA靶向。然后，我们将使用候选方法以及蛋白质组学和 CRISPR屏幕，以识别与这3英寸UTR域相互作用并调节的RNA结合蛋白 mb的mRNA靶向和局部翻译。其次，我们将测试MB的可能性 ESCRT mRNA的积累/翻译介导靶向MB的ESCRP复合物。第三，我们将测试 MBSOME依赖的特定mRNA是否转移，例如编码增殖调节剂的mRNA CENP-E有助于MBSOME诱导的细胞增殖。