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RNA递送至靶细胞，参与调控细胞迁移、发育与转移过程。为研究RNA包装进入外泌体的分子机制，本研究基于膜标志物CD63设计纯化方案，成功分离得到HEK293T细胞分泌的单一亚型外泌体。通过对该免疫分离获得的携带CD63的外泌体进行分析，我们鉴定出一组相较于细胞内源水平显著富集的微小RNA（miRNAs）。为探究外泌体生物发生与RNA包装的生化依赖条件，我们构建了无细胞反应体系，该体系可重现补充胞质的分离膜组分对miR-223的特异性选择性包裹过程。研究发现，RNA结合蛋白Y盒蛋白I（Y-box protein I，YBX1）可结合miR-223，且在该无细胞反应体系的miR-223分选过程中必不可少。此外，YBX1在HEK293T细胞向外泌体中分泌微小RNA的过程中发挥关键调控作用。