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.
Highlights
In contrast to the normal pathways of protein secretion, the processes by which unconventional cargoes are secreted have proved diverse and enigmatic
Proteins and RNAs can be secreted within vesicles that bud from the plasma membrane, as in the budding of enveloped viruses such as HIV, or within vesicles internalized into a multivesicular body (MVB) that fuses with the plasma membrane (Colombo et al, 2014)
Among the requirements for miRNA sorting in vitro, we found one RNA-binding protein, YBX1, which is a known constituent of exosomes secreted from intact cells (Ung et al, 2014; Buschow et al, 2010)
Summary
In contrast to the normal pathways of protein secretion, the processes by which unconventional cargoes are secreted have proved diverse and enigmatic. Secreted molecules may be externalized in a soluble form by translocation across various membranes. This may include direct translocation across the plasma membrane, or across an organelle membrane followed by fusion of the organelle with the plasma membrane (Zhang and Schekman, 2013). RNA is actively secreted into the medium of cultured cells and can be found in all bodily fluids enclosed within vesicles or bound up in ribonucleoprotin complexes, both forms of which are resistant to exogenous ribonuclease (Colombo et al, 2014; Arroyo et al, 2011; Mitchell et al, 2008). Extracellular vesicle-bound RNAs appear to be enriched in specific classes of RNAs, including small RNAs and microRNA (miRNA) (Skog et al, 2008; Valadi et al, 2007; Kosaka et al, 2010)
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