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Page 2 of 18 Happel et al. J Cancer Metastasis Treat 2020;6:32 I http://dx.doi.org/10.20517/2394-4722.2020.71

INTRODUCTION
Once thought to exist only within cells, RNA is now known to play a role in a variety of complex cellular
functions. Recent research has shown that RNA can be exported from cells and plays a role in the
[1,2]
molecular mechanisms of cell-to-cell communication . This paradigm-shifting observation launched the
field of extracellular RNA (exRNA) biology and represents a fundamental change in our understanding of
RNA in cell biology.

Extracellular RNA acts as a signalling molecule, traveling though body fluids carrying information from cell
to cell. Types of exRNA include both longer messenger RNA (mRNA) and long non-coding RNA (lncRNA),
as well as various types of small non-coding RNAs (ncRNAs). Non-coding RNAs can generally be broken
down into two groups, regulatory ncRNAs and housekeeping ncRNAs, as outlined in Table 1. Regulatory
ncRNAs include lncRNA, microRNA (miRNA), piwi-interacting RNA (piRNA), small interfering RNA
(siRNA), tRNA-derived fragments and Y RNA. Regulatory small ncRNAs have emerged as vital players in
various biological processes. They are known primarily for their role as regulators of gene expression at the
post-transcriptional level; however, they have a wide range of functions. Further information on individual
ncRNAs can be found in the review articles cited in Table 1. Housekeeping ncRNAs include ribosomal
RNA (rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), and small nucleolar RNA (snoRNA).
Housekeeping ncRNAs are highly abundant and are essential for cellular activities such as the translation
of RNA into proteins, and transcriptional splicing. The term exRNA includes many types of RNA.
Small non-coding exRNAs are often the focus of studies due to their abundance, ease of detection, and
regulatory function. MiRNA are of particular interest due to their role in post-transcriptional regulation
of gene expression. Changes in miRNA expression are associated with various pathological conditions and
[3]
dysregulation of miRNA expression is a hallmark of human cancer .

Extracellular RNA is secreted by all cell types and can be found in a variety of biofluids including plasma,
[4-7]
serum, breast milk, saliva, cerebrospinal fluid (CSF), bile, semen, and urine . While many ncRNAs are
found in human biofluids, miRNA, piRNA, snoRNA, tRNA-derived RNA fragments (tRF), and Y RNA
represent the most prominent types of exRNA found within various human biofluids (Figure 1 and Table
[4,8]
1, asterisks) . Carriers of exRNA include extracellular vesicles (EVs), ribonucleoprotein complexes
(RNPs), and lipoprotein complexes (LPPs). ExRNAs are either encased within extracellular vesicles, or, are
tightly associated with proteins to avoid degradation by RNAses. ExRNAs, in extracellular vesicles and/or
associated with protein complexes, can then be transferred from donor cells to recipient cells, where they
can elicit functional responses and regulate a number of biological processes [9,10] .

EVs, released by virtually all cell types, are small membrane-enclosed carriers of bioactive proteins, lipids,
[11]
and nucleic acids (including exRNAs) . Cells release a variety of EVs to transfer biological cargo to local
and distant recipient cells within the body to facilitate intercellular communication. The term extracellular
vesicles is broadly used for particles released from the cell that are delineated by a lipid bilayer, however,
there are multiple EVs subtypes which can be differentiated based on their size, biogenesis, release
[12]
pathways, cargo, and function . The main EV subpopulations include microvesicles (MVs), and exosomes.
MVs are approximately 100-1000 nm in size and are derived from outward blebbing of the plasma
[13]
membrane. Exosomes are approximately 30-100 nm vesicles of endosomal origin . The biogenesis of
exosomes begins with the formation of early endosomes by inward budding of the cell membrane, followed
by second inward budding of the endosomal membrane creating intraluminal vesicles (ILVs) and the larger
multivesicular bodies (MVBs). Fusion of the MVBs with the plasma membrane release ILVs as exosomes
into the extracellular milieu. Cytosolic constituents such as proteins and nucleic acids can be sorted into
both types of EVs as part of their respective biogenesis pathways [Figure 2].