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

项目1摘要： 细胞外囊泡（EV）携带多种RNA，包括编码和非编码RNA。这些 RNA具有影响细胞和组织表型的潜力。事实上，对于miRNAs， EV携带的miRNA在受体细胞中控制基因表达和功能的例子。RNA-Seq分析 已经证明了与细胞内容物相比，EV中某些RNA的特异性富集。可是 关于RNA被转运到EV中的具体机制知之甚少。 基于几项研究，目前的范式是RNA结合蛋白（RBP）负责 在EV中选择性和特异性地包含RNA。然而，这些RBP如何连接到蜂窝网络 将膜掺入脱落微泡（MV）或晚期内体衍生的外泌体中的方法是 未知一个值得注意的发现是，在EV中鉴定的许多RBP通常与内质网相关， 细胞中的内质网（ER），这表明ER在将这些部分转移到其他细胞器中的潜在作用。 此外，RNA诱导的沉默复合物组装在mRNA-核糖体复合物上， 与ER（粗糙ER），这表明了miRNA-RBP与ER以及随后的其他 膜。基于这些发现和我们的初步数据，我们假设ER-质膜 (PM)ER-多泡内体（MVE）膜接触位点（MCS）是RNA转移的关键 和RBP转化为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 在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 并确定MCS对RNA转移至受体细胞和CRC肿瘤生长的影响 和西妥昔单抗抗性。

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.