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Page 10 Cai et al. Extracell Vesicles Circ Nucleic Acids 2023;4:262-82 https://dx.doi.org/10.20517/evcna.2023.10

EV-mediated sRNA trafficking is also observed between mammalian hosts and their pathogens and
parasites, demonstrating the important role of EVs in cross-organismal sRNA trafficking. In the
aforementioned interaction between the nematode H. polygyrus and mice, H. polygyrus uses EVs to
[42]
transport miRNAs into the mouse cells . Similarly, human monocyte cells that deliver miRNAs into fungal
pathogen C. albicans also rely on EVs for this cross-kingdom RNAi . Within intra-organismal cellular
[122]
interactions in mammalian systems, EVs have been shown to traffic functional mRNA transcripts between
cells for protein translation in destination cells [128,129] . Human and mouse white blood cells produce EVs
containing hundreds of functional mRNA species . mRNA trafficking was a novel discovery in
[128]
mammalian cell-cell communication and revealed a new function of EV trafficking . Despite mRNA
[127]
intra-organismal trafficking via EVs, there have been no reports to date of functional mRNA cross-kingdom
trafficking in mammals, plants, or microbes.

SELECTIVE LOADING OF SMALL RNAS INTO EVS
Selective sorting of sRNAs into EVs has been previously reported and continues to be characterized in many
mammalian cell types. In human lymphocytes, hnRNPA2B1 protein was found to bind to sRNAs and load
[130]
sRNAs into exosomes depending on specific sequence motifs . A cleaved form of Rab-interacting
lysosomal protein (RILP) also contributes to exosomal miRNA loading depending on a conserved RNA
motif (AAUGC) in cultured HeLa cells . Another study in mouse neurons showed that miRNAs that
[131]
share a conserved four-nucleotide motif (GUAC) were selectively loaded into EVs .
[132]
Although selective RNA loading into EVs based on conserved motifs or RNA-binding proteins is
established in mammalian vesicle biology, research on selective loading is just beginning to be explored in
the study of plant and microbial EVs. In Arabidopsis, EV-sRNA profiling analysis revealed that the
enrichment of sRNA species in EVs does not correlate with their abundance inside the plant cell , which
[56]
suggests that these EV-enriched sRNAs are selectively loaded into EVs for transport. Proteomics analysis on
purified EVs isolated from B. cinerea infected Arabidopsis leaves revealed several RNA-binding proteins,
including Arabidopsis AGO1, DEAD-box RNA helicases (RH) 11, RH37, and RH52, which contribute to
EV selective sRNA loading . These RNA-binding proteins are localized in the membrane fractions inside
[56]
the cell and selectively bind with a subset of sRNAs that are 20-22 nt long, with the first 5’ nucleotide being
uracil. These RNA-binding proteins subsequently enter MVBs with associated sRNAs and are released
within exosomes into the apoplast. Another set of EV-localized RNA-binding proteins Annexin (ANN) 1
and ANN 2 bind sRNAs non-specifically. Furthermore, ago1, rh11/37, and ann1/2 mutants have reduced
[56]
levels of EV-enriched sRNAs in EVs . These results demonstrate that AGO1 and RH11/37/52 are
important for selective sRNA loading into EVs, and that although ANN1/2 are not involved in the selective
loading process, they are important for sRNA stabilization in EVs [Figure 1] . Similar EV-mediated cross-
[56]
species RNAi and RNA loading mechanisms exist in mammalian-parasite interactions. EVs of nematode
[42]
parasite H. polygyrus, which mediate cross-species RNAi within its mammalian host , contain an AGO
protein (exWAGO) that specifically binds to EV-enriched RNAs . Taken together, these results indicate
[133]
that AGO proteins are one of the components that are mainly responsible for selective RNA sorting into
EVs in plants and nematodes.

PATHOGEN-DERIVED EXTRACELLULAR VESICLES AND THEIR BIOLOGICAL
FUNCTIONS
EV secretion is a conserved process in all eukaryotic and prokaryotic cells [10,45] . Research has begun to
demonstrate the important role of microbial EVs in mediating the ability of bacteria and fungi to interact
with their hosts. Microbes, such as Gram-positive and Gram-negative bacteria, archaea, and fungi, have all
been shown to release EVs [134,135] . Gram-negative bacteria release 50 to 300 nm EVs by pinching off the