Abstract
The release and uptake of nano-sized extracellular vesicles (EV) is a highly conserved means of intercellular communication. The molecular composition of EV, and thereby their signaling function to target cells, is regulated by cellular activation and differentiation stimuli. EV are regarded as snapshots of cells and are, therefore, in the limelight as biomarkers for disease. Although research on EV-associated RNA has predominantly focused on microRNAs, the transcriptome of EV consists of multiple classes of small non-coding RNAs with potential gene-regulatory functions. It is not known whether environmental cues imposed on cells induce specific changes in a broad range of EV-associated RNA classes. Here, we investigated whether immune-activating or -suppressing stimuli imposed on primary dendritic cells affected the release of various small non-coding RNAs via EV. The small RNA transcriptomes of highly pure EV populations free from ribonucleoprotein particles were analyzed by RNA sequencing and RT-qPCR. Immune stimulus-specific changes were found in the miRNA, snoRNA, and Y-RNA content of EV from dendritic cells, whereas tRNA and snRNA levels were much less affected. Only part of the changes in EV-RNA content reflected changes in cellular RNA, which urges caution in interpreting EV as snapshots of cells. By comprehensive analysis of RNA obtained from highly purified EV, we demonstrate that multiple RNA classes contribute to genetic messages conveyed via EV. The identification of multiple RNA classes that display cell stimulation-dependent association with EV is the prelude to unraveling the function and biomarker potential of these EV-RNAs.
Highlights
Extracellular vesicles (EV) released by cells are considered as important mediators of intercellular communication [1, 2]
We isolated EV from cell culture supernatant of LPS-dendritic cells (DC), VitD3-DC, and control DC using differentialcentrifugation followed by density gradient separation, as previously published by our group (Supplementary Fig. 2A and [52])
Transmission electron microscopic analysis indicated that low-density fractions contained 100–150 nm-sized EV, whereas no EV were observed in high-density fractions enriched in protein complexes (Supplementary Fig. 2B)
Summary
Extracellular vesicles (EV) released by cells are considered as important mediators of intercellular communication [1, 2]. Exosomes are formed as intraluminal vesicles (ILVs) inside multi-vesicular endosomes (MVE) and are released upon fusion of the MVE with the plasma membrane, whereas microvesicles directly bud off from the plasma membrane [1]. These vesicle populations overlap in size and molecular composition, which currently hampers their discrimination based on biophysical or biochemical parameters [4,5,6]. The molecular composition of EV is often assumed to be a ‘snapshot’ of the producing cell, from which the tissue type or mutational status of the parental cell can be deduced [9, 11, 12]
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