Role of ER-membrane contacts in biogenesis of RNA-containing EVs

内质网膜接触在含 RNA EV 生物发生中的作用

• 批准号: 10544789
• 负责人: Alissa M Weaver
• 金额: $ 34.26万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-22 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10544789
• 关键词: Affect; Automobile Driving; Behavior; Binding; Biochemical; Biogenesis; Biological Models; Cell membrane; Cells; Cellular Membrane; Ceramides; Cetuximab; Cholesterol; Code; Collecting Cell; Communication; Complex; Data; Endoplasmic Reticulum; Endosomes; Engineering; Epidermal Growth Factor Receptor; Event; Fractionation; Gene Expression; Health; Human; In Vitro; KRAS2 gene; Large Intestine Carcinoma; Link; Lipids; Malignant Epithelial Cell; Mediating; Membrane; Messenger RNA; Methods; MicroRNAs; Organelles; Phenotype; Poly(A)+ RNA; Polyribosomes; Proteins; RNA; RNA Binding; RNA Transport; RNA-Binding Proteins; RNA-Induced Silencing Complex; Research; Resistance; Ribosomal Proteins; Ribosomes; Role; Rough endoplasmic reticulum; Route; Signal Transduction; Site; Small Nucleolar RNA; Testing; Tissues; Transfer RNA; Ubiquitination; Untranslated RNA; Vesicle; endosome membrane; exosome; extracellular; extracellular vesicles; gene function; in vivo; knock-down; late endosome; lipid transport; mRNA Translation; microvesicles; novel; overexpression; palmitoylation; protein complex; receptor; release of sequestered calcium ion into cytoplasm; segregation; small hairpin RNA; trait; transcriptome sequencing; tumor; tumor growth; tumor microenvironment; tumorigenesis; ubiquitin ligase

Project 1 Summary: Extracellular vesicles (EVs) carry a variety of RNAs, including both coding and noncoding RNAs. These RNAs have the potential to influence cell and tissue phenotypes. Indeed for miRNAs there are now many examples of EV-carried miRNA controlling gene expression and function in recipient cells. RNA-Seq analyses have demonstrated specific enrichment of some RNAs in EVs, compared to the cellular content. However, very little is known about the specific mechanisms by which RNA is transported into EVs. The current paradigm, based on several studies, is that RNA-binding proteins (RBPs) are responsible for the selective and specific inclusion of RNAs in EVs. However, how those RBPs connect to cellular membranes to be incorporated into shed microvesicles (MVs) or late endosome-derived exosomes is unknown. A notable finding is that many RBPs identified in EVs are typically associated with the endoplasmic reticulum (ER) in cells, suggesting a potential role for the ER in transfer of those moieties to other organelles. Furthermore, the RNA-induced silencing complex is assembled on mRNA-ribosome complexes associated with the ER (rough ER), suggesting a route for miRNA-RBP association with the ER and subsequently other membranes. Based on these findings and our preliminary data, we hypothesize that ER-plasma membrane (PM) and ER-multivesicular endosome (MVE) membrane contact sites (MCS) are critical for transfer of RNAs and RBPs into shed microvesicles and exosomes. We further hypothesize that signaling and lipid transfer events taking place at these contacts further regulate RNA transport into vesicles. We will test these hypotheses and determine the impact of MCS on transfer of RNAs to recipient cells and CRC tumor growth and cetuximab resistance.

项目1概要： 细胞外囊泡 (EV) 携带多种 RNA，包括编码 RNA 和非编码 RNA。这些 RNA 有可能影响细胞和组织表型。事实上，对于 miRNA，现在有很多 EV携带的miRNA控制受体细胞中基因表达和功能的例子。 RNA测序分析 已经证明，与细胞内容相比，EV 中的一些 RNA 具有特异性富集。然而，非常 人们对 RNA 转运至 EV 的具体机制知之甚少。 目前基于多项研究的范式是 RNA 结合蛋白 (RBP) 负责 用于选择性和特异性地将 RNA 包含在 EV 中。然而，这些 RBP 如何连接到蜂窝 要掺入脱落的微泡（MV）或晚期内体衍生的外泌体中的膜是 未知。一个值得注意的发现是，在 EV 中发现的许多 RBP 通常与内质 细胞中的网状结构（ER），表明 ER 在将这些部分转移到其他细胞器中具有潜在作用。 此外，RNA诱导的沉默复合物组装在相关的mRNA-核糖体复合物上。 与 ER（粗 ER）的关系，表明 miRNA-RBP 与 ER 以及随后的其他关联的途径 膜。根据这些发现和我们的初步数据，我们假设 ER 质膜 (PM) 和 ER-多囊内体 (MVE) 膜接触位点 (MCS) 对于 RNA 转移至关重要 和 RBP 进入脱落的微泡和外泌体。我们进一步假设信号传导和脂质转移 这些接触处发生的事件进一步调节 RNA 转运到囊泡中。我们将测试这些 假设并确定 MCS 对 RNA 转移至受体细胞和 CRC 肿瘤生长的影响 和西妥昔单抗耐药性。