Abstract
Extracellular vesicles (EVs) are important for intercellular communication and act as vehicles for biological material, such as various classes of coding and non-coding RNAs, a few of which were shown to selectively target into vesicles. However, protein factors, mechanisms, and sequence elements contributing to this specificity remain largely elusive. Here, we use a reporter system that results in different types of modified transcripts to decipher the specificity determinants of RNAs released into EVs. First, we found that small RNAs are more efficiently packaged into EVs than large ones, and second, we determined absolute quantities for several endogenous RNA transcripts in EVs (U6 snRNA, U1 snRNA, Y1 RNA, and GAPDH mRNA). We show that RNA polymerase III (pol III) transcripts are more efficiently secreted into EVs compared to pol II-derived transcripts. Surprisingly, our quantitative analysis revealed no RNA accumulation in the vesicles relative to the total cellular levels, based on both overexpressed reporter transcripts and endogenous RNAs. RNA appears to be EV-associated only at low copy numbers, ranging between 0.02 and 1 molecule per EV. This RNA association may reflect internal EV encapsulation or a less tightly bound state at the vesicle surface.
Highlights
Extracellular vesicles (EVs) are omnipresent lipid particles released by nearly every cell and are involved in cell–cell communication [1,2,3]
To cover a more comprehensive set of transcripts, we focused on polymerase III (pol III)-derived U6 snRNA and Y1 RNA, as well as on polymerase II (pol II)-derived U1 snRNA and GAPDH mRNA
An attractive approach to use EVs for therapeutic purposes involves loading the EVs with specific RNAs [39]
Summary
Extracellular vesicles (EVs) are omnipresent lipid particles released by nearly every cell and are involved in cell–cell communication [1,2,3]. As this already implies, EVs are not empty lipid bins, but rather represent transport vesicles, which are associated with various biomolecules. While most studies focus on the functionally well-characterized miRNAs [12,13,14], the advent of the RNA-Seq technologies allowed to analyze extremely low quantities of RNA, revealing different types of EV-associated RNA. The existence of mRNAs within vesicles could be demonstrated, transferable to and functionally active in recipient cells [20,21,22]
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