Unpacking extracellular vesicles: RNA cargo loading and function

Abstract

Extracellular vesicles (EVs) are a heterogeneous group of membrane-enclosed structures produced by prokaryotic and eukaryotic cells. EVs carry a range of biological cargoes, including RNA, protein, and lipids, which may have both metabolic significance and signalling potential. EV release has been suggested to play a critical role in maintaining intracellular homeostasis by eliminating unnecessary biological material from EV producing cells, and as a delivery system to enable cellular communication between both neighbouring and distant cells without physical contact. In this review, we give an overview of what is known about the relative enrichment of the different types of RNA that have been associated with EVs in the most recent research efforts. We then examine the selective and non-selective incorporation of these different RNA biotypes into EVs, the molecular systems of RNA sorting into EVs that have been elucidated so far, and the role of this process in EV-producing cells. Finally, we also discuss the model systems providing evidence for EV-mediated delivery of RNA to recipient cells, and the implications of this evidence for the relevance of this RNA delivery process in both physiological and pathological scenarios.

Keywords: EVs; RBPs; RNA; RNA loading; RNA‐binding proteins; delivery; extracellular vesicles; intercellular communication; loading; motifs; zipcodes.

FIGURE 1

Types of extracellular vesicle (EV) and extracellular RNA. Schematic diagram to represent the types of RNA found associated with extracellular vesicles (EVs), along with other co‐isolated extracellular RNAs. A number of extracellular complexes, such as RNAs and RNA binding proteins (RBPs), RNAs and Argonautes, or vault particles containing vault RNA (vtRNA) can also be found within/associated with EVs. Argonautes have also been reported in non‐membrane‐bound exomeres (Q. Zhang et al., ; H. Zhang et al., 2018). RBP‐RNA complexes can contain different RNAs such as messenger RNA (mRNA) or microRNA (miRNA) molecules. Although only ribosomal RNA (rRNA) and transfer RNA (tRNA) fragments are illustrated within EVs, other RNA fragments such as mRNA fragments, may also be present

FIGURE 2

Mechanisms influencing RNA EV‐incorporation. Schematic diagram summarising examples of mechanisms which have been proposed to influence RNA EV‐incorporation. RNA content may be determined in a passive manner, purely dependent on the local RNA concentrations at EV generation sites at a given point in time (a) (Tosar et al., 2015). The GC content or secondary structure associated with RNA sequence motifs (b) may also alter the affinity of associations between RNA molecules and lipid membranes or RNA binding proteins (RBPs) (c), and thus alter RNA concentrations at sites of EV formation, at the multivesicular body or at the plasma membrane (Janas et al., 2020, 2012). Examples of these RBPs, and the RNA biotypes that they associate with, are highlighted in the blue box. RBP and RNA interactions can also be modulated by RBP post‐translational modifications, such as SUMOylation and glycosylation (H. Lee et al., ; Villarroya‐Beltri et al., 2013). More recently, regulatory links have been proposed between EV biogenesis and lysosomal autophagy, named LC3‐dependent extracellular vesicle loading and secretion (LDELS) (Leidal et al., 2020) (d). Many RBPs associated with EV loading (HNRNPA2B1, HNRNPA1, HNRNPU, IGF2BP1, SYNCRIP, YBX‐1, FMR1) are known components of stress granules or P‐bodies (e), suggesting a possible connection between these RNA granules and loading of EV‐RNA (Leidal & Debnath, ; Liu et al., ; Markmiller et al., ; Wolozin & Ivanov, 2019). Modification of RNA, for example, via poly‐uridylation (polyU), poly‐adenylation (polyA) or methylation, for example, 5‐methylcytosine (m⁵C) and N6‐methyladenosine (m⁶A), may also be involved in determining EV packaging or cell retention (Koppers‐Lalic et al., 2014) (f). Coupling of EV‐RNA loading and upstream processes such as transcription and translation may also influence EV RNA loading (g). For example, a high abundance of polymerase‐III‐encoded transcripts has been observed within EVs (Hardy et al., ; Lefebvre et al., ; Mosbach et al., 2021). Within the cell RNAs become degraded via RNA decay (g), with features such as RNA motifs and RNA modifications influencing this process, and may generate preferentially stable fragments that become loaded into EVs a means of disposal (Van Balkom et al., 2015)

FIGURE 3

Functions of EV‐RNA. Schematic diagram to summarise the diversity of known mechanisms by which EV‐associated RNAs can modulate cellular activity. Exocytosis from EV‐producing cells may facilitate the disposal of excess or aberrant RNAs so as to not disrupt cell signalling pathways (a). EVs may also be internalised into recipient cells via endocytosis subsequently fusing with the endosome. EV RNAs present within the endosomal pathway may ultimately be degraded via the lysosome, as a way to outsource degradation to these recipient cells (Desdín‐Micó & Mittelbrunn, 2017) (b) RNA has also been shown to activate toll‐like receptors (TLRs) from within endosomes (c), for example, miR‐21 and miR‐29a activation of TLR‐7 and TLR‐8a (Fabbri et al., 2012). Endosomal escape has been suggested to be a limiting factor for functional cargo delivery (d), with only 10%–30% of internalised EVs estimated to release their cargo to the cytoplasm (Bonsergent et al., ; Joshi et al., 2020). Once RNAs have achieved endosomal escape, they can elicit effects from within the cytoplasm, such as translation of mRNA (Albanese et al., ; Valadi et al., 2007) (e), miRNA‐induced translational repression of mRNA (f), nuclear delivery (Hinger et al., ; Rimer et al., 2018) (g), or receptor activation, such as the pattern‐recognition receptor RIG‐1 (h) (Nabet et al., 2017)

FIGURE 4

Mechanisms of EV uptake. Delivery of EV RNAs to the recipient cell can occur via direct membrane fusion between the EV and the cell membrane of the recipient cell (a). An alternative EV uptake mechanism is via cellular contacts between EV producing and recipient cells, for example via nanotube structures or synapse formation (Haimovich et al., ; Mittelbrunn et al., 2011) (b). A common EV uptake mechanism is endocytosis (c), including clathrin‐mediated, phagocytosis, receptor‐mediated and micropinocytosis (Mulcahy et al., 2014)