Nowadays, there is no doubt that extracellular vesicles (EVs) are major contributors to cell-cell communication, although the underlying mechanisms have not yet been fully elucidated. Primarily, EVs are considered to deliver their diverse cargoes, from a donor to a recipient cell, in the context of intracellular communication and that EV-cell interaction is mediated via surface receptor/ligand interaction with subsequent signalling [1–3]. A particular focus of current research in this context is on the transfer of RNAs, especially in-light of the ongoing SARS-CoV-2 pandemic and the resulting boosted mRNA vaccine development . Since RNAs represent a rather unstable biomolecule, packaging is essential, which makes the use of elegant formulations of nanoparticles for the vaccines just mentioned obligatory . Interestingly, the use of naturally occurring EVs would be also feasible for this purpose, as possibly immune reactions can thus be weakened and more specific targeting can be ensured [4, 6, 7]. However, before we embark on this desirable goal and lose ourselves in the illusion that endogenous EVs could be the ultimate transfer vehicles for RNAs in the future, we need to extensively address the question of whether and how RNAs are packed into EVs. Which types of RNA are preferentially loaded into or associated with EVs? How abundant are they?
Certainly, all these questions can be extended to other cargos, especially proteins, for which absolute quantification also seems to be a sophisticated approach, but in this work, we would like to focus on RNAs.
Various RNA species in EVs: Abundance and functional relevance?
From their initial characterization, followed by ongoing studies on their function, the exact composition of EVs is still evolving [8, 9]. In addition to approaches of defining the individual EV subtypes in more detail with specific marker proteins, which are also controversially debated, the discussion about potential nucleic acid cargoes of EVs had already begun during the early phase of research. The concept of circulating nucleic acids (CNA), also referred to as extracellular nucleic acids (exNAs) has been established rather early, even before the first description of EVs in which cell-free circulating nucleic acids were detected in human serum . As EVs have emerged as the new “hot topic” in recent years, the number of reports on nucleic acids and EVs have also increased, especially those considering RNA as one of the (main) constituents of EVs. This ongoing development in the identification of new RNA subtypes in EVs is largely based on the progressive and increasingly sensitive next-generation sequencing (NGS) approaches. Thus, various RNA species in EVs could be comprehensively identified using high-throughput RNA-Seq and/or RT-qPCR approaches. These RNA species mainly include non-coding RNAs such as miRNAs, lncRNAs, snoRNAs, tRNAs, rRNAs, piRNAs,
Y RNAs, and circRNAs . In addition, fragments, as well as functionally active mRNAs, have also been detected in EVs . Based on this, the transfer of RNA via EVs became one of the most established and well-characterized functions of the latter mentioned. However, miRNAs in particular have attracted the attention of EV-associated RNAs in recent years, which is currently reflected by more than 4,700 listed publications in PubMed when searching for “extracellular vesicles” and “miRNA” (https://pubmed.ncbi.nlm.nih.gov/). The emphasis on the latter one is to some extent due to the well-established and attractive function of miRNAs in gene regulation. In general, it is nowadays still assumed that RNA is one of the major constituents inside EVs.
However, it should be taken into account that both, the terminology as well as the purification methods for the various EV subpopulations are still under development and in a constant state of optimization and validation flux, making a uniform standardization infeasible. We will probably never obtain a golden standard, too heterogeneous are the starting materials and research questions. Some features such as size, density, protein markers, and biogenesis may help in defining populations and their specific RNA cargo, although multiple features are present across populations [9, 13, 14]. As a result, it is very difficult to evaluate the individual studies that focused on RNA contents and their concentrations since a direct comparison is often not possible.
Strikingly, a provocative hypothesis has recently emerged: RNAs, here again in particular miRNAs, may be less abundant in EVs than initially assumed. Instead, miRNAs appear as stable extracellular RNAs in non-vesicular fractions [15, 16]. Based on an absolute quantification of EV-associated RNAs evidence emerges that only a few to very few molecules can be detected per vesicle [17, 18]. This refers in particular to the miRNAs, but also to all the other RNA species associated with EVs . Moreover, it makes no difference whether these are endogenous RNAs or derived from reporter constructs overexpressed in the cell of origin . The rationale for this change in the paradigm is due to improved purification methods, which clearly show that a direct correlation between the EV purification method and RNA yield can be observed  suggesting that previous results are rather related to EV-associated RNAs than to RNAs packaged into EVs. Whereas differential ultracentrifugation used to be the method of choice in early years of EV research, combinations of several methods such as differential ultracentrifugation and size exclusion chromatography and/or density gradient centrifugation are currently being favoured . This at least partially counteracts the co-purification of exRNAs, RNA binding protein (RNP) complexes and EVs, which results in lower RNA yield, since contaminating RNAs are no longer purified. In this context it is worth noting that in studies in which EVs were purified with high stringency, no RNA binding proteins (RBPs) could be identified (at least not those reported to be reported to be involved in RNA export into EVs) [8, 9]. Considering the dogma "RNA is never naked", the question now arises about how the RNAs are present in the vesicles. Is it sufficient that the vesicle itself protects RNA, and if so, how is the RNA exported from the cell into the EVs without RNA-binding proteins? Are the RNA binding proteins stripped off first? Although several mechanisms have already been linked to a selective release of RNAs, including sequence motifs and association with RBPs, the proposed mechanisms appear to be specific only to a well-defined subpopulation of RNAs rather than representing a general pathway [22, 23].
What does this mean for us? Do we need to rethink now about the enrichment and function of (mi)RNAs in EVs, which may not play a major role in EV biology?
Nevertheless, we should keep an open mind when studying extracellular RNAs. Especially for miRNA, which, even if they are not enriched in EVs, remain important and valuable established extracellular RNAs that play an important role in biomarker development. From our point of view, the ongoing evolution of sophisticated and state-of-the-art technologies for isolation and analysis by the EV community are mandatory to unravel all the upcoming questions. It might become true that RNAs INSIDE vesicles have been overestimated, but this fact does not exclude that RNA can still be associated WITH and/or ON vesicles, preserving a functional role. As an example, the idea of a corona enveloping EVs  seems very interesting in our opinion (Figure 1).
However, further characterization of this EV-corona will also require additional implementation in terms of purification in order to be able to obtain valid conclusions.
More recently, the possibility of (glycosylated) RNAs being directly anchored to the membrane and thus being exposed appears to be even more attractive, which could raise the scope of the efficacy of EV-associated RNAs to new heights. However, it remains at this point to be investigated in more detail .
Finally, despite all the controversy, we believe that extracellular RNAs represent an exciting area of basic, medical as well as applied science and highly appreciate and an increasing amount of research, doesn’t matter if it's in- or outside of EVs.
This work was funded by the Deutsche Forschungsgemeinschaft 416910386 - GRK 2573/1 (to E.P.), RTG2355 (to M.M.) and project Bi 316/20-1 within Research Unit FOR 5116 (to C.P.).