title: Y-box protein 1 is required to sort microRNAs into exosomes in cells and in a cell-free reaction.
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.
authors: Department of Plant and Microbial Biology, University of California, Berkeley, United States.
authors: Matthew J
authors: 0000-0001-9846-3051
authors: MJ
authors: Shurtleff
authors: Department of Plant and Microbial Biology, University of California, Berkeley, United States.
authors: Morayma M
authors: MM
authors: Temoche-Diaz
authors: Institute of Molecular Biology, University of Oregon, Eugene, United States.
authors: Kate V
authors: KV
authors: Karfilis
authors: Department of Molecular and Cellular Biology, Howard Hughes Medical Institute, University of California, Berkeley, United States.
authors: Sayaka
authors: S
authors: Ri
authors: Department of Molecular and Cellular Biology, Howard Hughes Medical Institute, University of California, Berkeley, United States.
authors: Randy
authors: 0000-0001-8615-6409
authors: R
authors: Schekman
mesh_terms: D002465:Cell Movement; D002474:Cell-Free System / Q000737:chemistry / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D055354:Exosomes / Q000737:chemistry / Q000378:metabolism*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D035683:MicroRNAs / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D060149:Tetraspanin 30 / Q000235:genetics / Q000378:metabolism; D051840:Y-Box-Binding Protein 1 / Q000235:genetics / Q000378:metabolism*
chemical_list: D007074:Immunoglobulin G; D008156:Luciferases; D035683:MicroRNAs; D060149:Tetraspanin 30; D051840:Y-Box-Binding Protein 1; C554586:CD63 protein, human; C525067:MIR223, human; C070856:YBX1 protein, human
keywords: Y-box; YBX1; biochemistry; cancer biology; exosome; extracellular vesicles; human; microRNA