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<p dir="ltr">Cells within our bodies communicate with each other through various mechanisms, one of which is through the use of small vesicles generically referred to as ‘extracellular vesicles’ (EVs). Notably, EVs are secreted by nearly all types of cells and harbor in them biomolecules that they derive from the parent cells. Our first study from the lab in the field of EV biology revealed that EVs from non-small cell lung cancer cells induce invasive phenotype in non-tumorigenic bronchial epithelial cells. Remarkably, RNA component of EVs stands out as a significant contributor to EV-related function. To determine specifics about the RNA subsets functional in the invasive phenotype in recipient cells, we performed an RNA sequencing analysis of total RNA from EVs of NSCLC and non-tumorigenic cells. Indeed, there were unique patterns of enrichment of RNA subsets in the EVs from the NSCLC cells. Amongst the RNAs uniquely enriched in Calu6 EVs, miR-10b, miR-100 and miR-155 were the most prominent. Interestingly, further studies leading to defining the cargo most crucial for driving invasive potential in non-tumorigenic cells, revealed the combinatorial impact of these three miRNAs. Our RNA sequencing analysis not only revealed unique patterns of enrichment of RNAs in NSCLC EVs, but also revealed something unexpected, that is the presence of miRNA subsets in EVs that are lost from NSCLC cells. We hypothesized that the export of miRNAs in EVs is a mechanism that cancer cells use to deplete themselves of specific subsets of miRNAs. To begin to delineate the pathway of export, we discovered a five-nucleotide RNA motif in the miRNAs enriched in NSCLC cell-derived EVs. Notably, this motif was not identified in EVs from non-tumorigenic cells. Interestingly, the motif was found to be necessary for the export of miRNAs into EVs. Not only that but the identified motif is also exported out into EVs through a mechanism that is specific to cancer cells. This further leads us to delineate the process of sequence-based export into EVs by identifying the cellular machinery that recognizes this motif in miRNAs and/or ‘marks’ them for export into EVs. To delve deeper into the understanding of the dynamics of RNA export into EVs, we successfully developed a method using flow cytometry to analyze the export of fluorescently labeled miRNAs by the cells into the EVs. The power of the developed tool will be used in fluorescence based CRISPR Cas9 screening to identify cellular features of miRNA export into EVs.</p>