Genome-wide interrogation of extracellular vesicle biology using barcoded miRNAs
Extracellular vesicles mediate transfer of biologically active molecules between neighboring or distant cells, and these vesicles may play important roles in normal physiology and the pathogenesis of multiple disease states including cancer. However, the underlying molecular mechanisms of their biog...
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doaj-e490d784dda64bbaba0b5e43cc646de72021-05-05T16:21:42ZengeLife Sciences Publications LtdeLife2050-084X2018-12-01710.7554/eLife.41460Genome-wide interrogation of extracellular vesicle biology using barcoded miRNAsAlbert Lu0Paulina Wawro1David W Morgens2Fernando Portela3https://orcid.org/0000-0003-0238-9251Michael C Bassik4Suzanne R Pfeffer5https://orcid.org/0000-0002-6462-984XDepartment of Biochemistry, Stanford University School of Medicine, Stanford, United StatesDepartment of Biochemistry, Stanford University School of Medicine, Stanford, United StatesDepartment of Genetics, Stanford University School of Medicine, Stanford, United StatesDepartment of Biochemistry, Stanford University School of Medicine, Stanford, United StatesDepartment of Genetics, Stanford University School of Medicine, Stanford, United StatesDepartment of Biochemistry, Stanford University School of Medicine, Stanford, United StatesExtracellular vesicles mediate transfer of biologically active molecules between neighboring or distant cells, and these vesicles may play important roles in normal physiology and the pathogenesis of multiple disease states including cancer. However, the underlying molecular mechanisms of their biogenesis and release remain unknown. We designed artificially barcoded, exosomal microRNAs (bEXOmiRs) to monitor extracellular vesicle release quantitatively using deep sequencing. We then expressed distinct pairs of CRISPR guide RNAs and bEXOmiRs, enabling identification of genes influencing bEXOmiR secretion from Cas9-edited cells. This approach uncovered genes with unrecognized roles in multivesicular endosome exocytosis, including critical roles for Wnt signaling in extracellular vesicle release regulation. Coupling bEXOmiR reporter analysis with CRISPR-Cas9 screening provides a powerful and unbiased means to study extracellular vesicle biology and for the first time, to associate a nucleic acid tag with individual membrane vesicles.https://elifesciences.org/articles/41460exosomemiRNAWnt signalingextracellular vesiclegenome wide screen |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Albert Lu Paulina Wawro David W Morgens Fernando Portela Michael C Bassik Suzanne R Pfeffer |
spellingShingle |
Albert Lu Paulina Wawro David W Morgens Fernando Portela Michael C Bassik Suzanne R Pfeffer Genome-wide interrogation of extracellular vesicle biology using barcoded miRNAs eLife exosome miRNA Wnt signaling extracellular vesicle genome wide screen |
author_facet |
Albert Lu Paulina Wawro David W Morgens Fernando Portela Michael C Bassik Suzanne R Pfeffer |
author_sort |
Albert Lu |
title |
Genome-wide interrogation of extracellular vesicle biology using barcoded miRNAs |
title_short |
Genome-wide interrogation of extracellular vesicle biology using barcoded miRNAs |
title_full |
Genome-wide interrogation of extracellular vesicle biology using barcoded miRNAs |
title_fullStr |
Genome-wide interrogation of extracellular vesicle biology using barcoded miRNAs |
title_full_unstemmed |
Genome-wide interrogation of extracellular vesicle biology using barcoded miRNAs |
title_sort |
genome-wide interrogation of extracellular vesicle biology using barcoded mirnas |
publisher |
eLife Sciences Publications Ltd |
series |
eLife |
issn |
2050-084X |
publishDate |
2018-12-01 |
description |
Extracellular vesicles mediate transfer of biologically active molecules between neighboring or distant cells, and these vesicles may play important roles in normal physiology and the pathogenesis of multiple disease states including cancer. However, the underlying molecular mechanisms of their biogenesis and release remain unknown. We designed artificially barcoded, exosomal microRNAs (bEXOmiRs) to monitor extracellular vesicle release quantitatively using deep sequencing. We then expressed distinct pairs of CRISPR guide RNAs and bEXOmiRs, enabling identification of genes influencing bEXOmiR secretion from Cas9-edited cells. This approach uncovered genes with unrecognized roles in multivesicular endosome exocytosis, including critical roles for Wnt signaling in extracellular vesicle release regulation. Coupling bEXOmiR reporter analysis with CRISPR-Cas9 screening provides a powerful and unbiased means to study extracellular vesicle biology and for the first time, to associate a nucleic acid tag with individual membrane vesicles. |
topic |
exosome miRNA Wnt signaling extracellular vesicle genome wide screen |
url |
https://elifesciences.org/articles/41460 |
work_keys_str_mv |
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