Obstacles and opportunities in the functional analysis of extracellular vesicle RNA - an ISEV position paper

Abstract

The release of RNA-containing extracellular vesicles (EV) into the extracellular milieu has been demonstrated in a multitude of different in vitro cell systems and in a variety of body fluids. RNA-containing EV are in the limelight for their capacity to communicate genetically encoded messages to other cells, their suitability as candidate biomarkers for diseases, and their use as therapeutic agents. Although EV-RNA has attracted enormous interest from basic researchers, clinicians, and industry, we currently have limited knowledge on which mechanisms drive and regulate RNA incorporation into EV and on how RNA-encoded messages affect signalling processes in EV-targeted cells. Moreover, EV-RNA research faces various technical challenges, such as standardisation of EV isolation methods, optimisation of methodologies to isolate and characterise minute quantities of RNA found in EV, and development of approaches to demonstrate functional transfer of EV-RNA in vivo. These topics were discussed at the 2015 EV-RNA workshop of the International Society for Extracellular Vesicles. This position paper was written by the participants of the workshop not only to give an overview of the current state of knowledge in the field, but also to clarify that our incomplete knowledge - of the nature of EV(-RNA)s and of how to effectively and reliably study them - currently prohibits the implementation of gold standards in EV-RNA research. In addition, this paper creates awareness of possibilities and limitations of currently used strategies to investigate EV-RNA and calls for caution in interpretation of the obtained data.

Keywords: Extracellular vesicles; RNA binding proteins; exosomes; function; mRNA; non-coding RNA; quantification; sorting.

Figure 1.

Schematic illustration of commonly used EV isolation techniques. (a) Differential centrifugation is the sequential pelleting of particles with decreasing sedimentation coefficients. Typically 2000 g is used to pellet large EVs, 10,000–20,000 g to pellet middle-sized EVs (green), and finally ~100,000 g to pellet the smallest EVs (different EV subpopulations are indicated in grey and orange). At these high g-forces, complexes of soluble proteins (black dots) may also sediment. (b) Lipids have a density that is approximately 1 g cm^–3, while proteins and RNA have a higher density (>1.3 g cm^–3). Therefore density gradients can be used to separate subpopulations of EVs with different ratio of lipids, RNA, and proteins. Moreover, these gradients can be used to purify vesicles away from soluble proteins, RNA, and protein–RNA complexes as the latter structures will not float at the same density as the lipid containing EVs. (c) Size exclusion chromatography separates particles based on their size, by trapping the smaller molecules (such as proteins and protein complexes) in the pores. The larger molecules (such as EVs) are too large to enter the pores and will elute first. (d) Precipitation of EV from cell culture medium or body fluids is based on volume-excluding polymers such as polyethylene glycol (PEG) with which biological materials such as proteins and EVs are precipitated from the solution. (e) (Immuno-)affinity capture isolates vesicles using beads coated with antibodies or proteins (such as heparin) with affinity for an EV transmembrane protein. Vesicles displaying the protein of interest will bind to the beads and can thereby be isolated from the vesicle-containing solution.

Figure 2.

Suggested mechanisms for EV-RNA sorting by RNA-binding proteins (RBPs). RNA may be packaged into EVs via active or passive mechanisms. RNA-binding proteins (RBPs) could bind intracellular RNAs bearing certain motifs or signals (structure, sequence or size) (1). Specific interaction between RNA/RBPs and endomembranes through docking receptors (2) or microtubule-docking receptors (3) may result in the local enrichment of RNA close to membrane compartments, thereby modulating their selective incorporation into EVs (4 and 5). Alternatively, RBPs may also be passively incorporated into EVs and protect their cargo in the extracellular space (6). Non-templated RNA modifications (e.g. uridylation) known to regulate RNA-turnover in cells (7), are also hypothesised to impact EV-RNA sorting by a still unknown mechanism. Upon viral infection, cellular stress, or miRNA-induced silencing, RNA can be selectively stored in cytoplasmic RNA granules (e.g. P/GW bodies) (8). This may balance their passive/active incorporation into EVs, either negatively by decreasing their soluble pool, or positively by interactions between GW-bodies and MVBs (9). The depicted processes may be tightly regulated by distinct signalling pathways (e.g. RAS, AKT) that trigger specific post-translational modification (PTMs) on RBPs or RNA-editing on transcripts, thereby affecting the stability and subcellular localisation of RNA/RBP complexes (10).

Figure 3.

Extracellular vesicle uptake and cargo delivery in recipient cells. EVs may release their cargo into the cytosol through direct fusion with the plasma membrane. Alternatively, EVs may be internalised via a variety of endocytic mechanisms, including clathrin-dependent endocytosis, clathrin-independent endocytosis, macropinocytosis and phagocytosis. Subsequently, EVs are transported into the cytoplasm in endocytic vesicles. These vesicles may proceed to scan the endoplasmatic reticulum (I), which has been reported to be a site for translation and RNA interference. EVs may fuse with endosomal membranes after acidification to release their RNA content (II), or be directed to lysosomes where they are degraded (III).

Figure 4.

Considerations for analysing the nature and function of EV-associated RNA. Overview of research questions aimed at unravelling the nature and function of EV-RNA and considerations in addressing these questions, as discussed at the 2015 ISEV workshop on EV-RNA.