1. Biochemistry and Chemical Biology
  2. Cancer Biology

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
https://doi.org/10.7554/eLife.19276
Share this article
Cite this article
  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
  2. Download BibTeX
  3. Download .RIS

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.
    http://dx.doi.org/10.5061/dryad.83420
    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.
    http://dx.doi.org/10.5061/dryad.83420

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

Version 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.

Metrics

  • 11,398
    Page views
  • 2,798
    Downloads
  • 417
    Citations

Article citation count generated by polling the highest count across the following sources: Scopus, Crossref, PubMed Central.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  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

Categories and tags

Research organism

Further reading

    1. Biochemistry and Chemical Biology
    2. Cell Biology

    Fluorescein-based sensors to purify human a-cells for functional and transcriptomic analyses

    Sevim Kahraman, Kimitaka Shibue ... Rohit N Kulkarni
    Tools and Resources

    Pancreatic a-cells secrete glucagon, an insulin counter-regulatory peptide hormone critical for the maintenance of glucose homeostasis. Investigation of the function of human a-cells remains a challenge due to the lack of cost-effective purification methods to isolate high-quality a-cells from islets. Here, we use the reaction-based probe diacetylated Zinpyr1 (DA-ZP1) to introduce a novel and simple method for enriching live a-cells from dissociated human islet cells with ~ 95% purity. The a-cells, confirmed by sorting and immunostaining for glucagon, were cultured up to 10 days to form a-pseudoislets. The a-pseudoislets could be maintained in culture without significant loss of viability, and responded to glucose challenge by secreting appropriate levels of glucagon. RNA-sequencing analyses (RNA-seq) revealed that expression levels of key a-cell identity genes were sustained in culture while some of the genes such as DLK1, GSN, SMIM24 were altered in a-pseudoislets in a time-dependent manner. In conclusion, we report a method to sort human primary a-cells with high purity that can be used for downstream analyses such as functional and transcriptional studies.

    1. Biochemistry and Chemical Biology
    2. Cell Biology

    Inositol pyrophosphate dynamics reveals control of the yeast phosphate starvation program through 1,5-IP8 and the SPX domain of Pho81

    Valentin Chabert, Geun-Don Kim ... Andreas Mayer
    Research Article

    Eukaryotic cells control inorganic phosphate to balance its role as essential macronutrient with its negative bioenergetic impact on reactions liberating phosphate. Phosphate homeostasis depends on the conserved INPHORS signaling pathway that utilizes inositol pyrophosphates and SPX receptor domains. Since cells synthesize various inositol pyrophosphates and SPX domains bind them promiscuously, it is unclear whether a specific inositol pyrophosphate regulates SPX domains in vivo, or whether multiple inositol pyrophosphates act as a pool. In contrast to previous models, which postulated that phosphate starvation is signaled by increased production of the inositol pyrophosphate 1-IP7, we now show that the levels of all detectable inositol pyrophosphates of yeast, 1-IP7, 5-IP7, and 1,5-IP8, strongly decline upon phosphate starvation. Among these, specifically the decline of 1,5-IP8 triggers the transcriptional phosphate starvation response, the PHO pathway. 1,5-IP8 inactivates the cyclin-dependent kinase inhibitor Pho81 through its SPX domain. This stimulates the cyclin-dependent kinase Pho85-Pho80 to phosphorylate the transcription factor Pho4 and repress the PHO pathway. Combining our results with observations from other systems, we propose a unified model where 1,5-IP8 signals cytosolic phosphate abundance to SPX proteins in fungi, plants, and mammals. Its absence triggers starvation responses.

    1. Biochemistry and Chemical Biology
    2. Cell Biology

    The FAM104 proteins VCF1/2 promote the nuclear localization of p97/VCP

    Maria Körner, Susanne R Meyer ... Alexander Buchberger
    Research Article

    The ATPase p97 (also known as VCP, Cdc48) has crucial functions in a variety of important cellular processes such as protein quality control, organellar homeostasis and DNA damage repair, and its de-regulation is linked to neuro-muscular diseases and cancer. p97 is tightly controlled by numerous regulatory cofactors, but the full range and function of the p97-cofactor network is unknown. Here, we identify the hitherto uncharacterized FAM104 proteins as a conserved family of p97 interactors. The two human family members VCP nuclear cofactor family member 1 and 2 (VCF1/2) bind p97 directly via a novel, alpha-helical motif and associate with p97-UFD1-NPL4 and p97-UBXN2B complexes in cells. VCF1/2 localize to the nucleus and promote the nuclear import of p97. Loss of VCF1/2 results in reduced nuclear p97 levels, slow growth and hypersensitivity to chemical inhibition of p97 in the absence and presence of DNA damage, suggesting that FAM104 proteins are critical regulators of nuclear p97 functions.