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.