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

TLDR

Exosomes are small vesicles secreted from metazoan cells that can transfer selected membrane proteins and small RNAs to target cells, influencing cell migration, development, and metastasis. The study aimed to investigate RNA packaging into exosomes by developing a CD63-based purification scheme and a cell‑free reaction that recapitulates miR‑223 enclosure in isolated membranes supplemented with cytosol. The authors isolated CD63‑positive exosomes from HEK293T cells and established a cell‑free system using isolated membranes and cytosol to model miR‑223 packaging. The study identified miRNAs enriched in CD63‑positive exosomes, showed that YBX1 binds and is essential for miR‑223 sorting in the cell‑free system, and demonstrated that YBX1 also promotes miRNA secretion in HEK293T cells.

Abstract

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.