RNA in extracellular vesicles
Kyoung Mi Kim
1,#
, Kotb Abdelmohsen
1
, Maja Mustapic
2
, Dimitrios Kapogiannis
2
, and
Myriam Gorospe
1
1
Laboratory of Genetics and Genomics, National Institute on Aging Intramural Research Program,
National Institutes of Health, Baltimore, MD 21224, USA
2
Laboratory of Neurosciences, National Institute on Aging Intramural Research Program, National
Institutes of Health, Baltimore, MD 21224, USA
Abstract
Cells release a range of membrane-enclosed extracellular vesicles (EVs) into the environment.
Among them, exosomes and microvesicles (collectively measuring 30-1000 nm in diameter) carry
proteins, signaling lipids, and nucleic acids from donor cells to recipient cells, and thus have been
proposed to serve as intercellular mediators of communication. EVs transport cellular materials in
many physiologic processes, including differentiation, stem cell homeostasis, immune responses,
and neuronal signaling. EVs are also increasingly recognized as having a direct role in
pathological processes, notably cancer and neurodegeneration. Accordingly, EVs have been the
focus of intense investigation as biomarkers of disease and prognostic indicators, and even
therapeutic tools. Here, we review the classes of RNAs present in EVs, both coding RNAs
(mRNAs) and noncoding RNAs (long noncoding RNAs, microRNAs, and circular RNAs). The
rising attention to EV-resident RNAs as biomarkers stems from the fact that RNAs can be detected
at extremely low quantities using a number of methods. To illustrate the interest in EV biology, we
discuss EV RNAs in cancer and neurodegeneration, two major age-associated disease processes.
Graphical abstract
#
Correspondence: Kyoung Mi Kim, Laboratory of Genetics and Genomics, National Institute on Aging, National Institutes of Health,
251 Bayview Blvd. Baltimore, MD 21224, USA, Tel: +1 410 558 8216; Fax: +1 410 558 8331, kyoungmi.kim@nih.gov.
CONFLICTS OF INTEREST
The authors have no conflicts of interest.
HHS Public Access
Author manuscript
Wiley Interdiscip Rev RNA
. Author manuscript; available in PMC 2018 July 01.
Published in final edited form as:
Wiley Interdiscip Rev RNA
. 2017 July ; 8(4): . doi:10.1002/wrna.1413.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
Keywords
Extracellular vesicle; exosome; microvesicle; microRNA; long noncoding RNA; circular RNA;
biomarker; gene therapy; drug delivery
INTRODUCTION
Extracellular Vesicles
Cells produce extracellular vesicles (EVs) of three main types, according to their size and
biogenesis: exosomes, microvesicles, and apoptotic bodies.
1-4
Microvesicles, measuring
0.2-1 μm in diameter, arise through budding of the plasma membrane and are therefore
enclosed by a fraction of plasma membrane.
5, 6
Apoptotic bodies are released from blebbing
of the plasma membrane of apoptotic cells and have diameters of 0.5-2 μm.
7, 8
Exosomes, by
contrast, arise from the endosomal pathway and form intracellular multivesicular bodies
(MVBs) which fuse to the plasma membrane and are secreted as vesicles measuring 0.04-0.1
μm. EVs were first described by Pan
et al
. and Johnstone
et al
. in 1983 in sheep reticulocytes
and were subsequently visualized using electron microscopy (EM)
9, 10, 11
EVs are secreted
from numerous cell types and have been isolated from a wide variety of human body fluids
such as blood, urine, saliva, and breast milk.
12-15
A variety of molecules have been identified in EVs, including DNA, RNA, bioactive lipids,
and proteins.
16
These molecules are protected by EV membranes from nucleases, proteases,
fluctuations in pH and osmolarity, and other environmental factors.
17-20
EV components can
be delivered from an originating cell to a recipient cell, whether the recipient cell is in the
vicinity (horizontal transfer) or in a distant tissue, and the transferred molecules are capable
of eliciting changes in function and gene expression in the recipient cell.
20-22
Since EVs are
found in easily accessible body fluids, particularly blood, they are attractive sources of
diagnostic and prognostic biomarkers.
23, 24
In addition, since EVs are derived from
intracellular material, they are being explored as packaging tools for the therapeutic delivery
of genetic material and drugs.
25-29
As we gain more information about EVs directed to
specific tissues and organs, such delivery of therapeutic molecules could be targeted with
high precision.
30
Given the rising interest in exploiting EVs in disease diagnosis and
treatment, there is urgency to determine comprehensively the tissues of origin of EVs, their
target tissues, and their molecular constituents. Towards the latter goal, the relatively low
abundance of EVs presents some challenges, but highly sensitive methods of RNA detection
developed in recent years [particularly RNA-sequencing (RNA-Seq) and reverse
transcription and quantitative PCR analysis (RT-qPCR)] afford accurate and quantitative
identification of RNAs, even if present in very low amounts. In this review, we discuss the
progress made by the RNA and EV communities to identify the pools of transcripts present
in exosomes and microvesicles.
DIVERSE RNAs IN EVs
The identification of RNAs in EVs has progressed immensely in recent years thanks to
technical advances in the detection of low-abundance, complex RNA samples. RNA pools in
Kim et al.
Page 2
Wiley Interdiscip Rev RNA
. Author manuscript; available in PMC 2018 July 01.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
EVs have been identified comprehensively using high-throughput RNA-Seq and many have
been validated using RT-qPCR analysis. These RNA populations include various protein-
coding transcripts (mRNAs) and many types of non-coding RNAs, including microRNAs
(miRNA), long noncoding RNAs (lncRNAs), circular RNAs (circRNAs), small nucleolar
RNA (snoRNAs), small nuclear RNAs (snRNAs), transfer RNA (tRNAs), ribosomal RNAs
(rRNAs), and piwi-interacting RNAs (piRNAs).
31
These RNAs can be transferred from
parent cells to recipient cells, where they can regulate or serve as templates for protein
production,
32, 33
although the relative abundance of full-length and fragmented transcripts in
EVs is not known at present because the analysis methods available (RNA-Seq and
microarray) identify relatively small RNA segments. In this review, we focus on mRNAs,
microRNAs, lncRNAs, and circRNAs identified in EVs.
Protein-coding RNA (mRNA)
Protein-coding RNAs are synthesized in the nucleus as pre-mRNAs and then typically
undergo splicing, modification of the ends, and export to the cytosol, where they function as
templates for protein translation. mRNAs have three basic segments, the 5′-untranslated
region (5′UTR), the coding region (CR, which encodes protein), and the 3′UTR. A number
of reports have identified full-length and sometimes fragmented mRNAs in EVs. For
example, early microarray analysis revealed that EVs (primarily exosomes) derived from
glioblastoma cells contained 27,000 mRNAs.
20
Interestingly, ~4,700 of these mRNAs were
only detected in EVs, not in cells, and >3,000 mRNAs were preferentially included (2,238
mRNAs) or excluded (1,188 mRNAs) from EVs compared with mRNAs found in cells. To
test whether mRNAs in glioblastoma-produced EVs were translated in recipient cells, the
authors expressed
Gaussia
luciferase
4
(
Gluc
) mRNA in glioblastoma cells and purified EVs
from the medium.
Gluc
mRNA encodes a luciferase protein that is secreted and emits
intense fluorescence. They then added these EVs to recipient human brain microvascular
endothelial cells (HBMVECs) and found that Gluc activity released by HBMVECs
increased continuously over the ensuing 24 h. These findings supported the view that the
cargo
Gluc
mRNA from the parent cell was translated in the recipient cell to generate a
functional protein. These results contribute to a substantial body of evidence that EV-
resident mRNAs can be translated in recipient cells.
Another study used microarrays to identify 13,000 mRNAs in EVs derived from MC/9 cells
(a mouse mast cell line).
34
Interestingly, 270 mRNAs were only detected in EVs and not in
cells. To test if the EV mRNAs could serve as a templates for the synthesis of functional
proteins, they used rabbit reticulocyte lysate as an
in vitro
translation system. Following the
completion of translation, two-dimensional polyacrylamide gel electrophoresis was
employed to identify mouse proteins (COX5B, HSPA8, SHMT1, LDH1, ZFP125, GPI1, and
RAD23B) from rabbit proteins by mass spectrometry analysis. The authors concluded that
many EV-resident mRNAs were translated efficiently.
In another model system, human central nervous system (CNS)-patrolling macrophages,
stimulation by β-amyloid peptide (Aβ) resulted in the secretion of exosomes containing a
number of cytokine mRNAs relevant to Alzheimer’s disease (AD) pathogenesis.
Interestingly, macrophages derived from older subjects generated higher levels of exosomal
Kim et al.
Page 3
Wiley Interdiscip Rev RNA
. Author manuscript; available in PMC 2018 July 01.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
IL6
,
TNF
, and
IL12
mRNAs, but did not exhibit differences in the levels of
IL8
,
IL1
, or
IL23
mRNAs. The authors proposed that cytokine mRNAs in exosomes may be a
mechanism for spreading neuroinflammation induced by Aβ peptide.
35
In sum, many
mRNAs residing in EVs can be translated and contribute to the protein expression programs
of recipient cells.
microRNA (miRNA)
MicroRNAs are small (~22-nt) non-coding, highly conserved, single-stranded RNAs found
both inside and outside of cells.
36, 37
The biosynthesis of microRNAs begins with the
transcription of a primary (pri-) microRNA that is processed by the RNase III DROSHA into
a ~70 nucleotide stem-loop transcript, the precursor (pre-)microRNA. XPO5 (Exportin-5)
then exports pre-microRNAs to the cytoplasm where they are processed by RNase III
DICER1 into mature ~22-nt microRNA duplexes;
37, 38
after unwinding the duplex, one
strand associates with AGO (Argonaute) proteins to form RNA-induced silencing complex
(RISC).
36, 38, 39
Alternative pathways for microRNA biogenesis have also been described.
40
Although microRNAs can affect gene transcription by influencing chromatin structure and
transcription, they are best known for eliciting gene silencing by lowering the stability
and/or translation of mRNAs with which they share partial complementarity, generally at the
mRNA 3′UTR.
36, 41, 42
By modulating gene expression programs in the cells in which they
are generated, microRNAs play a role in a wide range of biological processes, including
development, cell proliferation and differentiation, apoptosis, and immune regulation.
41
MicroRNAs can also participate in intercellular signaling. Many body fluids harbor
abundant, stable microRNAs which avoid nucleolytic degradation by associating with RNA-
binding proteins (RBPs) and high- and low-density lipoproteins, and by being encapsulated
in EVs.
43-45
With the discovery of microRNAs in EVs, many new functions and applications
have emerged – from new ways of cell-cell communication to potentially easy-access
biomarkers and, given the non-immunogenicity of EVs, possibly novel therapeutics.
19, 46-51
MicroRNAs uptaken via EVs might function as gene expression regulators in recipient cells,
but growing evidence from EVs in cancer and other processes has expanded the subset of
EV functions.
47, 52-55
Particularly surprising and exciting was the discovery that EV
microRNAs can act as ligands for Toll-like receptors (TLR) and induce immune responses
or inhibit macrophage activation by suppressing TLR signaling.
47, 56-59
It is still unclear
whether specific microRNAs are actively sorted into EVs, although some microRNAs
appear to be selectively targeted to EVs and several mechanisms for selective inclusion of
microRNAs have been proposed, as discussed in the section below.
Different microRNA profiles in patients and controls have been reported for many diseases,
implicating them in disease pathogenesis.
43, 46, 50, 60-63
The easy access and stability of EV-
microRNAs in biological fluids have created a niche for them as potential diagnostic
biomarkers. Even though most of such studies to-date come from cancer research, the brain
has the highest expression of tissue-specific microRNAs (70% of all reported microRNAs)
and thus microRNAs in brain EVs may also be particularly informative in neurological
disorders.
64, 65
Kim et al.
Page 4
Wiley Interdiscip Rev RNA
. Author manuscript; available in PMC 2018 July 01.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
The presence in EVs of misfolded proteins associated with neurodegenerative disorders [Aβ,
tau (MAPT), α-synuclein (SNCA), and prion proteins] prompted studies to test a role for
EVs in disease propagation.
66-69
The discovery of microRNAs in EVs expanded the search
for mechanisms of disease pathogenesis implicating dysregulation of endogenous
microRNAs.
66, 68, 70, 71
For example, the EV-resident microRNA miR-193b was implicated
in regulating the production of amyloid precursor protein (APP, the precursor of the
neurotoxic peptide Aβ), perhaps by repressing APP expression.
72
Interestingly, the levels of
soluble miR-193b in the blood of patients with MCI (mild cognitive impairment) and AD
were similar to those in controls, but EV miR-193b levels in the blood and cerebrospinal
fluid (CSF) of persons with MCI and AD was lower,
72
suggesting that miR-193b was
selectively excluded from these disease-associated populations. Sixteen microRNAs
implicated in AD were recovered from patients’ EVs, reaching a sensitivity and specificity
of 87% and 77% for predicting AD. Another set of seven EV microRNAs showed 83-89%
accuracy in predicting AD status and recent reviews showcase various microRNA signatures
for AD in different body fluids.
73
Six EV microRNAs have been associated with AD by
multiple groups; among them, miR191-5b in plasma and serum was particularly informative
in the diagnosis of AD.
74
Likewise, differential microRNA profiles were reported when
comparing CSF EVs from Parkinson’s disease (PD) and AD patients.
62
Age-related microRNA and TLR signaling dysregulation may contribute to other diseases of
aging, such as muscular and cardiovascular diseases.
75-78
Notably, sarcopenia in older
individuals could be associated with microRNA dysregulation, and changes in microRNA
signatures in muscle tissues have been observed with aging.
79, 80
The potential of EV microRNAs to change gene expression locally and distantly, together
with their non-immunogenic nature, further suggests that they hold great potential for
therapeutic applications.
46, 81, 82
An important caveat is the variable stoichiometry in
microRNAs contained in EVs.
83, 84
It is currently unknown whether all EVs in a population
contain the same amount of microRNAs or whether some vesicles are selectively loaded
with given microRNAs; this information is critical for therapeutic applications, since the
successful suppression of a given mRNA depends on the types and concentrations of
available microRNAs. Even though therapeutic applications of EV microRNAs are still in
their infancy, several successful studies
in vitro
and
in vivo
have been reported. In renal
fibrosis, EVs from mesenchymal stem cells were engineered to overexpress the microRNA
let-7c and were selectively targeted to damaged kidneys, where they successfully attenuated
kidney injury.
85
Similar approaches based on the EV-mediated delivery of microRNAs have
been undertaken for the treatment of cancer and liver disease.
86, 87
This field is expected to
boom with increasing knowledge of the targeted sorting, enrichment and packaging of
microRNAs into EVs.
Long noncoding RNA (lncRNA)
This class of noncoding RNAs, defined as being >200 nt, was identified almost three
decades ago,
88
but the advent of high-throughput RNA-Seq has begun to reveal the rich and
diverse functions of mammalian lncRNAs.
89-91
LncRNAs are involved in controlling a
Kim et al.
Page 5
Wiley Interdiscip Rev RNA
. Author manuscript; available in PMC 2018 July 01.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript