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Journal ArticleDOI

Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells

07 May 2007-Nature Cell Biology (Nature Publishing Group)-Vol. 9, Iss: 6, pp 654-659
TL;DR: It is shown that exosomes contain both mRNA and microRNA, which can be delivered to another cell, and can be functional in this new location, and it is proposed that this RNA is called “exosomal shuttle RNA” (esRNA).
Abstract: Exosomes are vesicles of endocytic origin released by many cells. These vesicles can mediate communication between cells, facilitating processes such as antigen presentation. Here, we show that exosomes from a mouse and a human mast cell line (MC/9 and HMC-1, respectively), as well as primary bone marrow-derived mouse mast cells, contain RNA. Microarray assessments revealed the presence of mRNA from approximately 1300 genes, many of which are not present in the cytoplasm of the donor cell. In vitro translation proved that the exosome mRNAs were functional. Quality control RNA analysis of total RNA derived from exosomes also revealed presence of small RNAs, including microRNAs. The RNA from mast cell exosomes is transferable to other mouse and human mast cells. After transfer of mouse exosomal RNA to human mast cells, new mouse proteins were found in the recipient cells, indicating that transferred exosomal mRNA can be translated after entering another cell. In summary, we show that exosomes contain both mRNA and microRNA, which can be delivered to another cell, and can be functional in this new location. We propose that this RNA is called "exosomal shuttle RNA" (esRNA).

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Citations
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01 Jan 2014
TL;DR: The definition of exosomes and other secreted extracellular vesicles, which mediating intercellular communication between different cell types in the body, and thus affecting normal and pathological conditions are focused on.
Abstract: In the 1980s, exosomes were described as vesicles of endosomal origin secreted from reticulocytes. Interest increased around these extracellular vesicles, as they appeared to participate in several cellular processes. Exosomes bear proteins, lipids, and RNAs, mediating intercellular communication between different cell types in the body, and thus affecting normal and pathological conditions. Only recently, scientists acknowledged the difficulty of separating exosomes from other types of extracellular vesicles, which precludes a clear attribution of a particular function to the different types of secreted vesicles. To shed light into this complex but expanding field of science, this review focuses on the definition of exosomes and other secreted extracellular vesicles. Their biogenesis, their secretion, and their subsequent fate are discussed, as their functions rely on these important processes.

3,321 citations


Cites background or methods from "Exosome-mediated transfer of mRNAs ..."

  • ...After the first description of nucleic acids in exosomes secreted by mast cells (Valadi et al. 2007), numerous groups have analyzed the presence of genetic material in EVs....

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  • ...Exosomes/Extracellular Vesicles and RNA A breakthrough in the field was made in 2007 when it was discovered that exosomes carry nucleic acids, namely mRNA and miRNA (Valadi et al. 2007)....

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  • ...As expected from the inside-in membrane orientation of all secreted EVs (i.e., cytosol inside and extracellular domains facing outside) (Figure 2), in most studies, mRNA are contained inside EVs, as shown by their resistance to RNAse digestion (Valadi et al. 2007)....

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  • ...…with Ca2+ ionophores has also been used in the literature to increase secretion of exosomes by the erythroleukemia cell line K562 (Savina et al. 2003), oligodendroglial cells (Krämer-Albers et al. 2007), DCs (Montecalvo et al. 2012), and mast cells (Raposo et al. 1997, Valadi et al. 2007)....

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  • ...From the time of their initial description in EVs (Ratajczak et al. 2006, Skog et al. 2008, Valadi et al. 2007), we have known that mRNA were not randomly secreted in exosomes, because different sequences were either preferentially secreted or, conversely, retained inside the cells....

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Journal ArticleDOI
TL;DR: The data show that exosome production, transfer and education of bone marrow cells supports tumor growth and metastasis, has prognostic value and offers promise for new therapeutic directions in the metastatic process.
Abstract: Tumor-derived exosomes are emerging mediators of tumorigenesis. We explored the function of melanoma-derived exosomes in the formation of primary tumors and metastases in mice and human subjects. Exosomes from highly metastatic melanomas increased the metastatic behavior of primary tumors by permanently 'educating' bone marrow progenitors through the receptor tyrosine kinase MET. Melanoma-derived exosomes also induced vascular leakiness at pre-metastatic sites and reprogrammed bone marrow progenitors toward a pro-vasculogenic phenotype that was positive for c-Kit, the receptor tyrosine kinase Tie2 and Met. Reducing Met expression in exosomes diminished the pro-metastatic behavior of bone marrow cells. Notably, MET expression was elevated in circulating CD45(-)C-KIT(low/+)TIE2(+) bone marrow progenitors from individuals with metastatic melanoma. RAB1A, RAB5B, RAB7 and RAB27A, regulators of membrane trafficking and exosome formation, were highly expressed in melanoma cells. Rab27A RNA interference decreased exosome production, preventing bone marrow education and reducing, tumor growth and metastasis. In addition, we identified an exosome-specific melanoma signature with prognostic and therapeutic potential comprised of TYRP2, VLA-4, HSP70, an HSP90 isoform and the MET oncoprotein. Our data show that exosome production, transfer and education of bone marrow cells supports tumor growth and metastasis, has prognostic value and offers promise for new therapeutic directions in the metastatic process.

3,076 citations

Journal ArticleDOI
TL;DR: Identification of extracellular Ago2–miRNA complexes in plasma raises the possibility that cells release a functional miRNA-induced silencing complex into the circulation, and reveals two populations of circulating miRNAs and suggest that circulating Ago2 complexes are a mechanism responsible for the stability of plasma mi RNAs.
Abstract: MicroRNAs (miRNAs) circulate in the bloodstream in a highly stable, extracellular form and are being developed as blood-based biomarkers for cancer and other diseases. However, the mechanism underlying their remarkable stability in the RNase-rich environment of blood is not well understood. The current model in the literature posits that circulating miRNAs are protected by encapsulation in membrane-bound vesicles such as exosomes, but this has not been systematically studied. We used differential centrifugation and size-exclusion chromatography as orthogonal approaches to characterize circulating miRNA complexes in human plasma and serum. We found, surprisingly, that the majority of circulating miRNAs cofractionated with protein complexes rather than with vesicles. miRNAs were also sensitive to protease treatment of plasma, indicating that protein complexes protect circulating miRNAs from plasma RNases. Further characterization revealed that Argonaute2 (Ago2), the key effector protein of miRNA-mediated silencing, was present in human plasma and eluted with plasma miRNAs in size-exclusion chromatography. Furthermore, immunoprecipitation of Ago2 from plasma readily recovered non–vesicle-associated plasma miRNAs. The majority of miRNAs studied copurified with the Ago2 ribonucleoprotein complex, but a minority of specific miRNAs associated predominantly with vesicles. Our results reveal two populations of circulating miRNAs and suggest that circulating Ago2 complexes are a mechanism responsible for the stability of plasma miRNAs. Our study has important implications for the development of biomarker approaches based on capture and analysis of circulating miRNAs. In addition, identification of extracellular Ago2–miRNA complexes in plasma raises the possibility that cells release a functional miRNA-induced silencing complex into the circulation.

2,900 citations


Cites background from "Exosome-mediated transfer of mRNAs ..."

  • ...Extracellular miRNAs have been proposed as a means of intercellular communication (6, 14, 16), and exosome-mediated transfer of miRNA between cells has been reported in cell culture (15) and from plasma-derived microvesicles to endothelial cells in the experimental setting (17)....

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Journal ArticleDOI
TL;DR: In this article, the authors describe principles of miRNA-mRNA interactions and proteins that interact with miRNAs and function in miRNA mediated repression, and discuss the multiple, often contradictory, mechanisms that miRNA have been reported to use, which cause translational repression and mRNA decay.
Abstract: MicroRNAs (miRNAs) are small noncoding RNAs that extensively regulate gene expression in animals, plants, and protozoa. miRNAs function posttranscriptionally by usually base-pairing to the mRNA 3′-untranslated regions to repress protein synthesis by mechanisms that are not fully understood. In this review, we describe principles of miRNA-mRNA interactions and proteins that interact with miRNAs and function in miRNA-mediated repression. We discuss the multiple, often contradictory, mechanisms that miRNAs have been reported to use, which cause translational repression and mRNA decay. We also address the issue of cellular localization of miRNA-mediated events and a role for RNA-binding proteins in activation or relief of miRNA repression.

2,762 citations


Cites background from "Exosome-mediated transfer of mRNAs ..."

  • ...Whether the latter phenomenon represents a specific way of elimination of miRISC components from the cell or is indicative of the miRNA-mediated intercellular communication (182) remains to be established....

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Journal ArticleDOI
TL;DR: Recent progress in understanding extracellular vesicle biology and the role of extrace cellular vesicles in disease is reviewed, emerging therapeutic opportunities are discussed and the associated challenges are considered.
Abstract: Within the past decade, extracellular vesicles have emerged as important mediators of intercellular communication, being involved in the transmission of biological signals between cells in both prokaryotes and higher eukaryotes to regulate a diverse range of biological processes. In addition, pathophysiological roles for extracellular vesicles are beginning to be recognized in diseases including cancer, infectious diseases and neurodegenerative disorders, highlighting potential novel targets for therapeutic intervention. Moreover, both unmodified and engineered extracellular vesicles are likely to have applications in macromolecular drug delivery. Here, we review recent progress in understanding extracellular vesicle biology and the role of extracellular vesicles in disease, discuss emerging therapeutic opportunities and consider the associated challenges.

2,507 citations

References
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Journal ArticleDOI
TL;DR: PicTar, a computational method for identifying common targets of micro RNAs, is presented and widespread coordinate control executed by microRNAs is suggested, thus providing evidence for coordinate microRNA control in mammals.
Abstract: MicroRNAs are small noncoding RNAs that recognize and bind to partially complementary sites in the 3' untranslated regions of target genes in animals and, by unknown mechanisms, regulate protein production of the target transcript. Different combinations of microRNAs are expressed in different cell types and may coordinately regulate cell-specific target genes. Here, we present PicTar, a computational method for identifying common targets of microRNAs. Statistical tests using genome-wide alignments of eight vertebrate genomes, PicTar's ability to specifically recover published microRNA targets, and experimental validation of seven predicted targets suggest that PicTar has an excellent success rate in predicting targets for single microRNAs and for combinations of microRNAs. We find that vertebrate microRNAs target, on average, roughly 200 transcripts each. Furthermore, our results suggest widespread coordinate control executed by microRNAs. In particular, we experimentally validate common regulation of Mtpn by miR-375, miR-124 and let-7b and thus provide evidence for coordinate microRNA control in mammals.

4,660 citations


"Exosome-mediated transfer of mRNAs ..." refers background in this paper

  • ...Hypothetically, the 121 miRNAs that we have found in the mast cell exosomes may interfere with 24,000 mRNAs as it has been suggested that each species may interact with up to 200 mRNA...

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Journal ArticleDOI
TL;DR: It is demonstrated by immunoelectron microscopy that the limiting membrane of MIICs can fuse directly with the plasma membrane, resulting in release from the cells of internal MHC class II-containing vesicles, suggesting a role for exosomes in antigen presentation in vivo.
Abstract: Antigen-presenting cells contain a specialized late endocytic compartment, MIIC (major histocompatibility complex [MHC] class II-enriched compartment), that harbors newly synthesized MHC class II molecules in transit to the plasma membrane. MIICs have a limiting membrane enclosing characteristic internal membrane vesicles. Both the limiting membrane and the internal vesicles contain MHC class II. In this study on B lymphoblastoid cells, we demonstrate by immunoelectron microscopy that the limiting membrane of MIICs can fuse directly with the plasma membrane, resulting in release from the cells of internal MHC class II-containing vesicles. These secreted vesicles, named exosomes, were isolated from the cell culture media by differential centrifugation followed by flotation on sucrose density gradients. The overall surface protein composition of exosomes differed significantly from that of the plasma membrane. Exosome-bound MHC class II was in a compact, peptide-bound conformation. Metabolically labeled MHC class II was released into the extracellular medium with relatively slow kinetics, 10 +/- 4% in 24 h, indicating that direct fusion of MIICs with the plasma membrane is not the major pathway by which MHC class II reaches the plasma membrane. Exosomes derived from both human and murine B lymphocytes induced antigen-specific MHC class II-restricted T cell responses. These data suggest a role for exosomes in antigen presentation in vivo.

2,978 citations

Journal ArticleDOI
TL;DR: The results indicate that exosome isolation may provide an efficient first step in biomarker discovery in urine and identify numerous protein components of MVBs and of the endosomal pathway in general.
Abstract: Urine provides an alternative to blood plasma as a potential source of disease biomarkers. One urinary biomarker already exploited in clinical studies is aquaporin-2. However, it remains a mystery how aquaporin-2 (an integral membrane protein) and other apical transporters are delivered to the urine. Here we address the hypothesis that these proteins reach the urine through the secretion of exosomes [membrane vesicles that originate as internal vesicles of multivesicular bodies (MVBs)]. Low-density urinary membrane vesicles from normal human subjects were isolated by differential centrifugation. ImmunoGold electron microscopy using antibodies directed to cytoplasmic or anticytoplasmic epitopes revealed that the vesicles are oriented "cytoplasmic-side inward," consistent with the unique orientation of exosomes. The vesicles were small (<100 nm), consistent with studies of MVBs and exosomes from other tissues. Proteomic analysis of urinary vesicles through nanospray liquid chromatography-tandem mass spectrometry identified numerous protein components of MVBs and of the endosomal pathway in general. Full liquid chromatography-tandem MS analysis revealed 295 proteins, including multiple protein products of genes already known to be responsible for renal and systemic diseases, including autosomal dominant polycystic kidney disease, Gitelman syndrome, Bartter syndrome, autosomal recessive syndrome of osteopetrosis with renal tubular acidosis, and familial renal hypomagnesemia. The results indicate that exosome isolation may provide an efficient first step in biomarker discovery in urine.

1,941 citations

Journal ArticleDOI
01 Jul 1983-Cell
TL;DR: The fate of the transferrin receptor during in vitro maturation of sheep reticulocytes has been followed using FITC- and 125I-labeled anti-transferrin-receptor antibodies and it can be shown that at 0 degree C or in phosphate-buffered saline the rate of vesicle release is less than that at 37 degrees C in culture medium.

1,543 citations


"Exosome-mediated transfer of mRNAs ..." refers background in this paper

  • ...Many cells have the capacity to release exosomes, including reticulocyte...

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Journal ArticleDOI
01 May 2006-Leukemia
TL;DR: ES-MV isolated from murine ES cells in serum-free cultures significantly enhanced survival and improved expansion of murine HPC, and upregulated the expression of early pluripotent and early hematopoietic stem cells in these cells.
Abstract: Membrane-derived vesicles (MV) are released from the surface of activated eucaryotic cells and exert pleiotropic effects on surrounding cells. Since the maintenance of pluripotency and undifferentiated propagation of embryonic stem (ES) cells in vitro requires tight cell to cell contacts and effective intercellular signaling, we hypothesize that MV derived from ES cells (ES-MV) express stem cell-specific molecules that may also support self-renewal and expansion of adult stem cells. To address this hypothesis, we employed expansion of hematopoietic progenitor cells (HPC) as a model. We found that ES-MV (10 microg/ml) isolated from murine ES cells (ES-D3) in serum-free cultures significantly (i) enhanced survival and improved expansion of murine HPC, (ii) upregulated the expression of early pluripotent (Oct-4, Nanog and Rex-1) and early hematopoietic stem cells (Scl, HoxB4 and GATA 2) markers in these cells, and (iii) induced phosphorylation of MAPK p42/44 and serine-threonine kinase AKT. Furthermore, molecular analysis revealed that ES-MV express Wnt-3 protein and are selectively highly enriched in mRNA for several pluripotent transcription factors as compared to parental ES cells. More important, this mRNA could be delivered by ES-MV to target cells and translated into the corresponding proteins. The biological effects of ES-MV were inhibited after heat inactivation or pretreatment with RNAse, indicating a major involvement of protein and mRNA components of ES-MV in the observed phenomena. We postulate that ES-MV may efficiently expand HPC by stimulating them with ES-MV expressed ligands (e.g., Wnt-3) as well as increase their pluripotency after horizontal transfer of ES-derived mRNA.

1,464 citations