Defining the parameters for sorting of different RNA cargo into Extracellular vesicles

Abstract Extracellular vesicles (EVs) are small particles that are released by cells and mediate cell-cell communication by transferring bioactive molecules. RNA cargo of EVs, including coding and non-coding RNAs, can change the behavior of recipient cells, affecting processes like gene expression, proliferation, and apoptosis. Circular RNAs (CircRNA) are a newly appreciated class of regulatory RNAs that are stable, resistant to degradation and have been shown to be enriched in EVs. They play key roles in gene regulation and are also emerging as promising biomarkers for disease diagnosis. While microRNAs (miRNAs) are the most well studied RNA cargo of EVs, very little is known about the mechanisms of enrichment of circRNAs as well as long linear RNAs. Here, we take a comprehensive genome-wide approach to investigate the role of GC%, size, exon count, structuredness and coding potential, in the sorting and enrichment of circular and long linear RNAs into EVs. We found that size and structuredness had a significant role in enriching RNAs into EVs which was consistent across all classes of RNAs. Furthermore, we found that structuredness could explain the relative enrichment of circRNAs over their linear counterparts. These results were validated on existing public databases of circular and linear RNAs in EVs. By identifying and analyzing these factors, we aim to better understand the complex mechanisms behind EV-mediated RNA transfer and its impact on cell communication in both health and disease. This mechanistic understanding of RNA enrichment in EVs is crucial for engineering EVs with selective RNA cargo.