Introduction to Extracellular Vesicles: Biogenesis, RNA Cargo Selection, Content, Release, and Uptake

Abstract

Extracellular vesicles are a heterogeneous group of membrane-limited vesicles loaded with various proteins, lipids, and nucleic acids. Release of extracellular vesicles from its cell of origin occurs either through the outward budding of the plasma membrane or through the inward budding of the endosomal membrane, resulting in the formation of multivesicular bodies, which release vesicles upon fusion with the plasma membrane. The release of vesicles can facilitate intercellular communication by contact with or by internalization of contents, either by fusion with the plasma membrane or by endocytosis into "recipient" cells. Although the interest in extracellular vesicle research is increasing, there are still no real standards in place to separate or classify the different types of vesicles. This review provides an introduction into this expanding and complex field of research focusing on the biogenesis, nucleic acid cargo loading, content, release, and uptake of extracellular vesicles.

Keywords: Biogenesis; Cargo selection; Exosomes; Extracellular vesicles; Microvesicles; RNA.

Fig. 1

Molecular mechanisms of ESCRT-dependent and -independent MVB biogenesis. Multiple biogenesis machineries have been described for generating ILVs in MVBs. a ESCRT-dependent MVB biogenesis requires the ESCRT protein and ESCRT-associated proteins (ALIX, TSG101, Chmp4, and SKD1) to form MVBs containing CD63, MHC II, ubiquitinated proteins and KFERQ-containing proteins. b Three ESCRT-independent pathways are controlled by different proteins: 1 heparanase and ARF6/PLD2, associated with the presence of syntenin-1, syndecan, and CD63 in exosomes; 2 nSMase, in which the exosomes are enriched with PLP, CD63, CD81, and TSG101 [Components in image derived from Servier Medical Art Powerpoint image bank (Servier 2016)]

Fig. 2

Molecular machineries of EV release. a Proteins involved in controlling the fusion of MVBs with the outer membrane to the plasma membrane, resulting in release of exosomes. Five different machineries have been described so far; 1 RAB11 and RAB35 facilitate the fusion of MVBs to the plasma membrane, releasing exosomes containing PLP, Wnt, flotillin, and TfR; 2 RAB27A and RAB27B promote release of exosomes loaded with CD63, TSG101, and ALIX; 3 RAB7-dependent release yields release of exosomes harboring ALIX, synthenin, and syndecan; 4 DGKα protein is implicated in release of exosomes carrying LAMP1, CD63, and Fas ligand; and 5 VAMP7 regulates the membrane fusion associated with release of acetylcholinesterase-containing exosomes release. b EV released via the outward budding and fission of the plasma membrane controlled by different proteins and extracellular signaling results in release of MVs with a distinct protein profile. Three pathways have been described including markers found in released MVs: a ARRDC1, TSG101, and VSP4 are responsible for the shedding of MVs containing TSG101 and ARRDC1; b hypoxia following expression of RAB22A via HIF, characterizes the secretion of EVs carrying TGM2; and c the ARF6, PLD, ERK, and MLCK cascade induces release of EVs containing gelatinases, ARF6, MHC-I, β1-integrin, VAMP3, and MT1MMP. [Components in image derived from Servier Medical Art Powerpoint image bank (Servier 2016)]

Fig. 3

RNA loading mechanisms and RNA species found in EVs. a A graphical representation of the different RNA species found in EVs including mRNA, miRNA, tRNA, rRNA, vault RNA, circRNA, Y RNA, lncRNA, and sncRNA. b Packaging of RNA within the lipid bilayer membrane is thought to protect it from RNase digestion once released into the extracellular environment. c Different mechanisms of mRNA and miRNA loading into EV as shown in the left panel include: the enrichment of miRNA in EVs due to cellular stress; hnRNPA2B1 binding to GAGG motif present miRNA; 3′ end uridylation of miRNAs/increasing nSMase2 activity resulting in miRNA loading; the abundance of miRNA target mRNA transcripts in the cell and the binding of miRNA to lipids associated with EVs. Loading of mRNA or mRNA fragments is based on the presence of zipcode sequence and association with miR1289. [Components in image derived from Servier Medical Art Powerpoint image bank (Servier 2016)]