Data from: Y-box protein 1 is required to sort microRNAs into exosomes in cells and in a cell-free reaction

Exosomes are small vesicles that are secreted from metazoan cells and may convey selected membrane proteins and small RNAs to target cells for the control of cell migration, development and metastasis. To study the mechanisms of RNA packaging into exosomes, we devised a purification scheme based on the membrane marker CD63 to isolate a single exosome species secreted from HEK293T cells. Using immunoisolated CD63-containing exosomes we identified a set of miRNAs that are highly enriched with respect to their cellular levels. To explore the biochemical requirements for exosome biogenesis and RNA packaging, we devised a cell-free reaction that recapitulates the species-selective enclosure of miR-223 in isolated membranes supplemented with cytosol. We found that the RNA-binding protein Y-box protein I (YBX1) binds to and is required for the sorting of miR-223 in the cell-free reaction. Furthermore, YBX1 serves an important role in the secretion of miRNAs in exosomes by HEK293T cells.

外泌体（Exosomes）是一类由多细胞动物细胞分泌的小型囊泡，可将特定膜蛋白与小型RNA转运至靶细胞，以调控细胞迁移、发育及转移过程。为研究RNA包装进入外泌体的机制，我们基于膜标记物CD63设计了纯化方案，以分离HEK293T细胞分泌的单一外泌体亚型。通过免疫分离携带CD63的外泌体，我们鉴定出一组相较于细胞内水平显著富集的微小RNA（miRNAs）。为探究外泌体生物发生与RNA包装的生化需求，我们构建了无细胞反应体系，可重现分离得到的膜组分在补充胞质溶胶后对miR-223的亚型选择性包裹过程。我们发现，RNA结合蛋白Y盒结合蛋白1（YBX1）可结合miR-223，且在该无细胞反应体系中是miR-223分选所必需的。此外，YBX1在HEK293T细胞分泌包裹于外泌体中的miRNAs过程中发挥重要作用。