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      By contrast, MVs range from a few nanometers to a few microns in diameter and derive from outward budding of the plasma membrane (Witwer et al. 2013; Kalluri 2016). In some lipids and phosphatidylserine they are enriched.

      Apoptotic bodies are released by apoptotic cells. They are the largest EVs, with a diameter of 1 to 5 μm, and contain several intracellular fragments, cellular organelles, membranes, and cytosolic contents (van der Pol et al. 2012; Cufaro et al. 2019). However, in practice it remains difficult to distinguish between the different subtypes of EVs. Therefore the ISEV recommends, by consensus, using the general term “EV” in the nomenclature (Gould and Raposo 2013; Thery et al. 2018). In this chapter we use this term to refer to all the subtypes of vesicles present in the extracellular space, alongside small EVs (sEVs) for exosomes.

      Additional components are found in EVs, including a wide variety of genetic molecules such as DNA and coding or non-coding RNAs (Nawaz et al. 2018).

      Components of Exosomes (EVs)

      RNAs

      Most of the RNA present in normal cells is ribosomal RNA, which is reported to account for 80–85% of total cellular RNA. Among its subunits, the 28S and 18S have the highest abundance; therefore, when electrophoresis of total RNA extracted from mammalian cells is performed, the two main bands represent the 28S and 18S subunits (Figure 3.2; see Imbeaud et al. 2005).

      Figure 3.2 Gel electrophoresis of mouse liver total RNA (left panel) and mouse serum EV-associated RNAs (right panel). Black triangles represent size markers.

      Both mRNAs and small RNAs in normal cells have low abundance by comparison with ribosomal RNAs. Small RNAs include small nuclear RNAs (snRNAs), miRNAs, small nucleolar RNAs (snoRNAs), and piwi-interacting RNAs (piRNAs) (Watson et al. 2019).

      In contrast, small RNAs, especially miRNAs, are enriched in sEVs (Figure 3.2; see Valadi et al. 2007). MicroRNAs (miRNAs) were originally discovered in Caenorhabditis elegans and are found in most eukaryotes, including humans and mice. These molecules are 21–25 base (nt)-long single-stranded RNAs that are involved in the post-transcriptional regulation of gene expression in eukaryotes (Lee et al. 1993; Ambros 2004). To date, 1974 hairpin precursors of miRNAs and 2654 mature miRNA sequences have been annotated in the human genome (Kozomara et al. 2019). The latest information about miRNAs is available in the miRBase database (http://www.mirbase.org). Each miRNA binds to its target mRNA with incomplete homology, generally recognizes the 3ʹUTR of the target gene, and destabilizes the target mRNA or represses its translation by translation inhibition (Macfarlane and Murphy 2010).

      Transcriptional repression mediated by miRNAs is known to play an important role in a wide variety of biological processes such as normal development, teratogenicity, cell proliferation and differentiation, apoptosis, and cancer formation (Alberti and Cochella 2017; Gueta et al. 2010; Mor et al. 2014; Macfarlane and Murphy 2010).

      The standard miRNA production process consists of the following five steps: (1) transcriptional synthesis of primary miRNA (pri-miRNA); (2) cleavage of the pri-miRNA in the nucleus, to produce precursor miRNA (pre-miRNA); (3) nuclear export of the pre-miRNA; (4) cleavage of the pre-miRNA in the cytoplasm to produce miRNA duplexes; and (5) maturation of the miRNA.

These processes are collectively referred to as “miRNA processing” (see Figure 3.3).

      1 Transcriptional synthesis of primary miRNA (pri-miRNA). The first miRNA precursor transcripts are produced in the nucleus through the transcription of the miRNA genes, primarily by RNA polymerase II and III, the same polymerases that transcribe those genes that are translated into proteins and polIII-driven repeat sequences (Lee et al. 2004; Borchert et al. 2006). Primary transcripts containing 60–70 nt RNA hairpin structures are called pri-miRNAs (Han et al. 2006). Most mammalian miRNAs are located within intergenic regions or within the introns of protein-encoding genes and non-coding RNAs. Less commonly, miRNA precursors may be located in exons of transcripts and in antisense transcripts.

      2 Cleavage of pri-miRNA in the nucleus to produce precursor miRNA (pre-miRNA). The ribonuclease III endonuclease (RNase III), Drosha, along with DGCR8 and multiple cofactors, cleaves the hairpin base of the pri-miRNA in the nucleus to produce a 60–70-base pre-miRNA with a hairpin structure, which is an intermediate (Denli et al. 2004; Gregory et al. 2004; Han et al. 2004; Lee et al. 2003; Lee et al. 2002).

      3 Nuclear export of pre-miRNA. Subsequent pre-miRNA processing occurs in the cytoplasm. The pre-miRNA is mainly transported from the nucleus to the cytoplasm by nucleocytoplasmic transporter containing exportin-5 (XPO5) and Ran-GTP, which prevents its degradation in the nucleus and facilitates its translocation into the cytoplasm (Bohnsack Czaplinski, Gorlich 2004; Okada et al. 2009; Yi et al. 2003; Zeng and Cullen 2004).

      4 Cleavage of pre-miRNA in the cytoplasm to produce miRNA duplexes. The pre-miRNA undergoes a second cleavage event, whichis mediated by another RNase III endonuclease, Dicer, with its cofactor TRBP. The resulting small RNA is a 21-to-24-base miRNA duplex (miRNA-5p/miRNA-3p) (Hutvagner et al. 2001; Chendrimada et al. 2005).

      5 Maturation of miRNA. A double-stranded miRNA duplex is incorporated into the Ago protein, and only one side of the RNA strand (the miRNA strand, guide strand, or mature miRNA) finally forms a stable complex with the Ago protein, forming an RNA-induced silencing complex (RISC) (Kwak and Tomari 2012; Iwasaki et al. 2010; Hammond et al. 2000). Finally, this single-stranded mature miRNA acts as a guide for gene expression regulation. RISC binds to its target mRNA, which has a sequence that is partially complementary to the miRNA incorporated into the RISC, and generally suppresses the output of the target mRNA to the protein (Jonas and Izaurralde 2015).

      Figure 3.3 Canonical miRNA biogenesis pathway.

To date, there have been several reports on the mechanism of EV capture of miRNAs. Those miRNAs whose expression levels increase in T cells upon activation are not significantly increased in exosomes (Villarroya-Beltri et al. 2013). A similar trend was observed for mRNAs, which suggests that miRNA and mRNA loading into exosomes is not a passive process. Several miRNAs were more highly represented in EVs than in cells, for example miR-575, miR-451, miR-125a-3p, miR-198, miR-601 and miR-887. Analysis of these expression profiles revealed that miRNAs that are preferentially sorted to EVs contain specific short motifs, called “EXO motifs.” The sumoylated heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) controls the loading of specific miRNAs into EVs by binding to these motifs. It was also demonstrated that HnRNPA2B1 could bind to an RNA trafficking sequence (RTS) of myelin basic protein (MBP) mRNA with a length of 21 nt and regulate mRNA trafficking to axons in neural cells (Munro et al. 1999). Sumoylation of HnRNPA2B1 is also required for the binding of RNA to HnRNPA2B1. Thus at least HnRNPA2B1 contributes to the loading of miRNAs into EVs (Villarroya-Beltri et al. 2013).

      Proteins

After EVs are released from cells, EV-associated miRNAs will be delivered to recipient cells through the body fluids. Surface proteins on EVs help EVs to first adhere to target cells, which is a fundamental step for EV-recipient cell communication. Tetraspanins are thought to have roles in adhesion, motility, signal transduction, and cell activation; and they are highly abundant on the exosome surface (Figure 3.4). These tetraspanins include CD9, CD53, CD63, CD81, and CD82; they may contribute to the spatial assembly of factors for antigen recognition and may partially dictate the signal induced by EVs (Simons and Raposo 2009; Mathivanan et al. 2010). In fact, the treatment of recipient cells with antibodies against CD81 or CD9 can reduce the uptake of EVs by recipient cells, which suggests that tetraspanins

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