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

  1. Matthew J Shurtleff
  2. Morayma M Temoche-Diaz
  3. Kate V Karfilis
  4. Sayaka Ri
  5. Randy Schekman  Is a corresponding author
  1. University of California, Berkeley, United States
  2. University of Oregon, United States
  3. Howard Hughes Medical Institute, University of California, Berkeley, United States

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.

Data availability

The following data sets were generated
    1. Shurtleff M
    2. Karfilis K
    3. Temoche-Diaz M
    4. Ri S
    5. Schekman R
    (2016) HEK293T cell small RNA-seq
    Available at Dryad Digital Repository under a CC0 Public Domain Dedication.
    1. Shurtleff M
    2. Karfilis K
    3. Temoche-Diaz M
    4. Ri S
    5. Schekman R
    (2016) Exosomes from HEK293T cells small RNA-seq reads
    Available at Dryad Digital Repository under a CC0 Public Domain Dedication.

Article and author information

Author details

  1. Matthew J Shurtleff

    Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9846-3051
  2. Morayma M Temoche-Diaz

    Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
  3. Kate V Karfilis

    Institute of Molecular Biology, University of Oregon, Eugene, United States
    Competing interests
    No competing interests declared.
  4. Sayaka Ri

    Department of Molecular and Cellular Biology, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
  5. Randy Schekman

    Department of Molecular and Cellular Biology, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States
    For correspondence
    schekman@berkeley.edu
    Competing interests
    Randy Schekman, EIC, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8615-6409

Funding

Howard Hughes Medical Institute (Investigator)

  • Randy Schekman

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Timothy W Nilsen, Case Western Reserve University, United States

Publication history

  1. Received: June 30, 2016
  2. Accepted: August 24, 2016
  3. Accepted Manuscript published: August 25, 2016 (version 1)
  4. Version of Record published: October 3, 2016 (version 2)

Copyright

© 2016, Shurtleff et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Matthew J Shurtleff
  2. Morayma M Temoche-Diaz
  3. Kate V Karfilis
  4. Sayaka Ri
  5. Randy Schekman
(2016)
Y-box protein 1 is required to sort microRNAs into exosomes in cells and in a cell-free reaction
eLife 5:e19276.
https://doi.org/10.7554/eLife.19276
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