Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells
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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TL;DR: It is shown here that miRNAs are present in human plasma in a remarkably stable form that is protected from endogenous RNase activity and established the measurement of tumor-derived mi RNAs in serum or plasma as an important approach for the blood-based detection of human cancer.
Abstract: Improved approaches for the detection of common epithelial malignancies are urgently needed to reduce the worldwide morbidity and mortality caused by cancer. MicroRNAs (miRNAs) are small (≈22 nt) regulatory RNAs that are frequently dysregulated in cancer and have shown promise as tissue-based markers for cancer classification and prognostication. We show here that miRNAs are present in human plasma in a remarkably stable form that is protected from endogenous RNase activity. miRNAs originating from human prostate cancer xenografts enter the circulation, are readily measured in plasma, and can robustly distinguish xenografted mice from controls. This concept extends to cancer in humans, where serum levels of miR-141 (a miRNA expressed in prostate cancer) can distinguish patients with prostate cancer from healthy controls. Our results establish the measurement of tumor-derived miRNAs in serum or plasma as an important approach for the blood-based detection of human cancer.
7,296 citations
Cites background from "Exosome-mediated transfer of mRNAs ..."
...Exosomes are 50- to 90-nm (24), membrane-bound particles that have been reported to be abundant in plasma (25) and that have recently been shown (in cell culture studies) to contain miRNAs (26)....
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TL;DR: This review focuses on the characterization of EVs and on currently proposed mechanisms for their formation, targeting, and function.
Abstract: Cells release into the extracellular environment diverse types of membrane vesicles of endosomal and plasma membrane origin called exosomes and microvesicles, respectively. These extracellular vesicles (EVs) represent an important mode of intercellular communication by serving as vehicles for transfer between cells of membrane and cytosolic proteins, lipids, and RNA. Deficiencies in our knowledge of the molecular mechanisms for EV formation and lack of methods to interfere with the packaging of cargo or with vesicle release, however, still hamper identification of their physiological relevance in vivo. In this review, we focus on the characterization of EVs and on currently proposed mechanisms for their formation, targeting, and function.
6,141 citations
Cites background from "Exosome-mediated transfer of mRNAs ..."
...A major breakthrough was the demonstration that the cargo of EVs included both mRNA and miRNA and that EVassociated mRNAs could be translated into proteins by target cells (Ratajczak et al., 2006; Valadi et al., 2007)....
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...Many RNAs that were isolated with EVs were found to be enriched relative to the RNA profiles of the originating cells (Ratajczak et al., 2006; Valadi et al., 2007; Skog et al., 2008; Nolte-’t Hoen et al., 2012a), indicating that RNA molecules are selectively incorporated into EVs....
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TL;DR: This Review summarizes the current understanding of the mechanistic aspects of microRNA-induced repression of translation and discusses some of the controversies regarding different modes of micro RNA function.
Abstract: MicroRNAs constitute a large family of small, approximately 21-nucleotide-long, non-coding RNAs that have emerged as key post-transcriptional regulators of gene expression in metazoans and plants. In mammals, microRNAs are predicted to control the activity of approximately 30% of all protein-coding genes, and have been shown to participate in the regulation of almost every cellular process investigated so far. By base pairing to mRNAs, microRNAs mediate translational repression or mRNA degradation. This Review summarizes the current understanding of the mechanistic aspects of microRNA-induced repression of translation and discusses some of the controversies regarding different modes of microRNA function.
4,973 citations
TL;DR: Extracellular vesicles are now considered as an additional mechanism for intercellular communication, allowing cells to exchange proteins, lipids and genetic material.
Abstract: Extracellular vesicles are a heterogeneous group of cell-derived membranous structures comprising exosomes and microvesicles, which originate from the endosomal system or which are shed from the plasma membrane, respectively They are present in biological fluids and are involved in multiple physiological and pathological processes Extracellular vesicles are now considered as an additional mechanism for intercellular communication, allowing cells to exchange proteins, lipids and genetic material Knowledge of the cellular processes that govern extracellular vesicle biology is essential to shed light on the physiological and pathological functions of these vesicles as well as on clinical applications involving their use and/or analysis However, in this expanding field, much remains unknown regarding the origin, biogenesis, secretion, targeting and fate of these vesicles
4,241 citations
TL;DR: Tumour-derived microvesicles may provide diagnostic information and aid in therapeutic decisions for cancer patients through a blood test by incorporating an mRNA for a reporter protein into them, and it is demonstrated that messages delivered by microvesicle are translated by recipient cells.
Abstract: Glioblastoma tumour cells release microvesicles (exosomes) containing mRNA, miRNA and angiogenic proteins. These microvesicles are taken up by normal host cells, such as brain microvascular endothelial cells. By incorporating an mRNA for a reporter protein into these microvesicles, we demonstrate that messages delivered by microvesicles are translated by recipient cells. These microvesicles are also enriched in angiogenic proteins and stimulate tubule formation by endothelial cells. Tumour-derived microvesicles therefore serve as a means of delivering genetic information and proteins to recipient cells in the tumour environment. Glioblastoma microvesicles also stimulated proliferation of a human glioma cell line, indicating a self-promoting aspect. Messenger RNA mutant/variants and miRNAs characteristic of gliomas could be detected in serum microvesicles of glioblastoma patients. The tumour-specific EGFRvIII was detected in serum microvesicles from 7 out of 25 glioblastoma patients. Thus, tumour-derived microvesicles may provide diagnostic information and aid in therapeutic decisions for cancer patients through a blood test.
4,118 citations
Cites background from "Exosome-mediated transfer of mRNAs ..."
...It has been shown that microvesicles can transfer some of their contents to other cell type...
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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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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
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
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.
Abstract: 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. Vesicles containing peptides that comigrate with the transferrin receptor on polyacrylamide gels are released during incubation of sheep reticulocytes, tagged with anti-transferrin-receptor antibodies. Vesicle formation does not require the presence of the anti-transferrin-receptor antibodies. Using 125I-surface-labeled reticulocytes, it can be shown that the 125I-labeled material which is released is retained by an immunoaffinity column of the anti-transferrin-receptor antibody. Using reticulocytes tagged with 125I-labeled anti-transferrin-receptor antibodies to follow the formation of vesicles, 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. There is selective externalization of the antibody-receptor complex since few other membrane proteins are found in the externalized vesicles. The anti-transferrin-receptor antibodies cause redistribution of the receptor into patches that do not appear to be required for vesicle formation.
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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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