Highly purified EVs isolated from human cell lines display a small number of substantially (~ 1000 fold) enriched miRNAs that differ from one cell line to another. In spite of the small number of such species, no single RNA sorting sequence is evident. In order to explore the mechanism of RNA sorting, we established a cell-free reaction that reproduces the selective incorporation of synthetic, mature miRNAs (miR223 and miR122) into vesicles formed in a reaction containing membranes and cytosol from mechanically disrupted HEK293 cells. The sorting reaction requires both membrane and cytosol and is stimulated by hydrolysable ATP and incubation at a physiologic temperature. Using biotinlyated derivatives of two different miRNAs, we found different sets of RNA binding proteins incorporated along with each species, among which the proteins Ybx1 and Lupus La are required to sort mir223 and miR122, respectively. EVs also contain more abundant major species of small RNA including full-length tRNA, Y-RNA and vault RNA, and each requires the Ybx1 protein for selective sorting into exosomes secreted by cells and into vesicles in the cell-free reaction. tRNAs in EVs appear to have a distinct chemical modification that is much less abundant in tRNAs in HEK293 cells. This modification may be involved in tRNA sorting or in the function of tRNA transported to a target cell. Shurtleff MJ, Temoche-Diaz MM, Karfilis KV, Ri S, Schekman R (2016). Y-box protein 1 is required to sort microRNAs into exosomes in cells and in a cell-free reaction. Elife. Aug 25;5. pii: e19276. doi: 10.7554/eLife.19276. PMID: 27559612 Shurtleff, M., Yao, J., Qin, Y, Nottingham, R., Temoche-Diaz, M., Schekman, R and Lambowitz, A. (2017) A broad role for YBX1 in defining the small non-coding RNA composition of exosomes PNAS 2017 October, 114 (43) E8987-E8995. https://doi.org/10.1073/pnas.1712108114 Shurtleff, M., Temoche-Diaz, M. and Schekman, R. (2018) Extracellular Vesicles and Cancer: Caveat Lector. Ann. Rev. Cancer Biology https://doi.org/10.1146/annurev-cancerbio-030617-050519