Shurtleff Matthew J, Temoche-Diaz Morayma M, Karfilis Kate V, Ri Sayaka, Schekman Randy
Department of Plant and Microbial Biology, University of California, Berkeley, United States.
Institute of Molecular Biology, University of Oregon, Eugene, United States.
Elife. 2016 Aug 25;5:e19276. doi: 10.7554/eLife.19276.
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.
外泌体是后生动物细胞分泌的小囊泡，可能将特定的膜蛋白和小RNA传递给靶细胞，以控制细胞迁移、发育和转移。为了研究RNA包装到外泌体中的机制，我们设计了一种基于膜标记物CD63的纯化方案，以分离从HEK293T细胞分泌的单一外泌体种类。使用免疫分离的含CD63的外泌体，我们鉴定了一组相对于其细胞水平高度富集的miRNA。为了探索外泌体生物发生和RNA包装的生化要求，我们设计了一种无细胞反应，该反应概括了在补充有胞质溶胶的分离膜中miR-223的物种选择性包封。我们发现RNA结合蛋白Y盒蛋白I（YBX1）在无细胞反应中与miR-223的分选结合并是其必需的。此外，YBX1在HEK293T细胞外泌体中miRNA的分泌中起重要作用。
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