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Extracellular Vesicles for Nucleic Acid Delivery
Published in Yashwant Pathak, Gene Delivery, 2022
Md Meraj Anjum, Dulla Naveen Kumar, Aiswarya Chaudhuri, Sanjay Singh, Ashish Kumar Agrawal
Apart from proteins and lipids, EVs are also comprised of RNA species. The EVs derived from mammalian cells are extensively scrutinized for the availability of RNA species, provided with latest methods of sequencing. The RNA includes messenger RNAs, non-coding RNAs, microRNA, transfer RNA, ribosomal RNA, small nuclear RNA, small nucleolar RNA, small cytoplasmic RNA, Y-RNA, and vault RNA [55]. Apart from RNAs, the mammalian EVs are also comprised of certain degraded products. However, the relative availability of various RNAs vary between donor cells and extravesicular cells. For instance, EVs derived from dendritic cells showed enhanced levels of vault RNA, SRPRNA, and Y-RNA as compared to the parent cells [73]. Overexpressed cellular miRNAs, like miR92a-1 and let-7b, were found to be in low concentrations in EVs, while highly expressed EV derived miRNAs, like miR-223, miR-142, and miR-93, were expressed at a lesser concentration in the parent cells. Such an instance implies that RNAs are sorted selectively into the extracellular vesicles during their biosynthesis. Nevertheless, the bacterial OMVs showed a smaller amount of expressed RNAs. It has been observed that the huge range of RNAs, like transfer RNA, transfer-messenger RNAs, ribosomal RNA, signal recognition particle-RNA, and 6S RNA, were found in EVs released from bacteria [62]. These findings are somewhat similar to the finding obtained from that of extracellular vesicles released from the mammalian cell, where the distinctive sorting of distinct RNAs has been unveiled. The detailed summary of protein, lipid components, and RNAs found in exosomes is given in Table 3.2.
How does an RNA selfie work? EV-associated RNA in innate immunity as self or danger
Published in Journal of Extracellular Vesicles, 2020
Yu Xiao, Tom Driedonks, Kenneth W. Witwer, Qian Wang, Hang Yin
To be sure, EV association may not be needed to trigger responses. In addition to being loaded into EVs [17], non-coding RNA species from MCF-7 cells, mainly dimers of tRNA-Gly-GCC halves and ribosomal RNA, are also released in non-EV fractions [18]. In this form, they can activate primary bone-marrow-derived dendritic cells and induce the release of IL-1β. It was hypothesized that these non-EV-associated RNAs may function as an immune surveillance mechanism, by which immune cells may sense damaged or dying cells (Figure 1). Furthermore, RNP-associated Y-RNA has also been shown to activate various RNA sensors [19].
Y-RNA subtype ratios in plasma extracellular vesicles are cell type- specific and are candidate biomarkers for inflammatory diseases
Published in Journal of Extracellular Vesicles, 2020
Tom A.P. Driedonks, Sanne Mol, Sanne de Bruin, Anna-Linda Peters, Xiaogang Zhang, Marthe F.S. Lindenbergh, Boukje M. Beuger, Anne-Marieke D. van Stalborch, Thom Spaan, Esther C. de Jong, Erhard van der Vries, Coert Margadant, Robin van Bruggen, Alexander P.J. Vlaar, Tom Groot Kormelink, Esther N.M. Nolte-‘T Hoen
EV in plasma are likely derived from multiple cell types. It is currently unknown whether a broad range of blood-related cell types has the capacity to release EV containing full length Y-RNA into the extracellular space. To investigate this, we isolated EV from in vitro cultures of human red blood cells (RBC), neutrophils, peripheral blood mononuclear cells (PBMC, which consist of T cells, B cells, and monocytes), and endothelial cells (HUVEC). We additionally prepared EV from platelets since these are known to be abundantly present in blood plasma [60]. We aimed to compare the number of EV released by these different cell types within a similar time frame. Because primary neutrophils are short-lived in culture [61], EV from all different blood-related cell types were collected after a short culturing period of 2–4 h. EV were purified by differential centrifugation followed by density gradient ultracentrifugation. By using high-resolution flow cytometric quantification of EV [50,51], we observed that all cells released EV within the given culture period, although the number of released EV per cell strongly differed between cell types (Figure 3(a)). Irrespective of these differences in released EV, we used RT-qPCR to assess whether full length Y-RNAs could be detected in EV from the selected blood cells (Figure 3(b)). Y-RNA subtypes Y1, Y3 and Y4 were abundantly detected in EV from all cell types. Y5 levels were substantially lower and were excluded in subsequent computational analyses because of the low reliability with which such low-abundance transcripts can be quantified. Across the different cell types, we found a statistically significant log-linear relationship between Y-RNA abundance and EV number, which indicates that cells that release more EV also release more Y-RNA (Figure 3(c)). Furthermore, we observed remarkable differences in the relative abundance of Y-RNA subtypes in EV from various cell types. Notably, neutrophil-derived EV were unique in their high levels of Y4 and low levels of Y3. In contrast, PBMC derived EV and platelet-derived EV contained high levels of both Y4 and Y3, but low levels of Y1. To further explore these cell-specific Y-RNA profiles, we calculated abundance-ratios between each of the Y-RNA subtypes, which we defined as the log2fold difference between individual Y-RNA subtypes (Figure 3(d)). Based on these calculations, we conclude that EV from neutrophils uniquely displayed high Y4/Y3 and negative Y3/Y1 ratios. PBMC- and platelet-EV were characterized by high Y3/Y1 ratios but Y4/Y3 ratios around zero. In EV from HUVEC and RBC, on the contrary, none of the Y-RNA-subtype ratios deviated significantly from zero. Overall, our results indicate that blood-related cell types differ in the amount of EV-associated Y-RNA they release. In addition, our data illustrate that EV released by different blood cells vary in Y-RNA subtype composition and therefore have a specific “Y-RNA signature”.