Fine-tuning of the innate immune response by microRNAs.

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REVIEW
Fine-Tuning of the Innate Immune Response by
microRNAs
Michael P Gantier, Anthony J Sadler and Bryan RG Williams
MicroRNAs (miRNAs) are emerging as potent regulators of many biological processes, including cellular differentiation and
disease. Recently, miRNA has been directly involved in innate immunity and transduction signalling by Toll-like receptors and
the ensuing cytokine response. In this review, we present an overview of what is currently known of the involvement of miRNA
and RNA interference components in the fine-tuning of innate immune responses.
Immunology and Cell Biology (2007) 0, 000–000. doi:10.1038/sj.icb.7100091
Keywords: innate immunity; microRNAs; RNA interference
Small RNAs (19–25 nt) play key gene-regulatory roles in many
eukaryotic lineages, including fungi, plants and animals. They were
first discovered 20 years ago in Caenorhabditis elegans.1,2 Two small
RNAs, lin-4 and let-7, were observed to alter temporal development.1,2
The lin-4 and let-7 RNAs are processed from a hairpin formed within
their respective transcripts.3,4 To date, 112 small RNAs formed from
hairpin structures have been identified in C. elegans,5 establishing a
class of endogenous RNAs called microRNAs (miRNAs) that are now
known to be critical regulators of numerous biological functions
throughout different species. This review focuses on the intricate
relationships between miRNAs and innate immunity in mammals.
miRNA BIOGENESIS
A number of classes of small RNAs, besides miRNA, have been
identified. The best characterized of these are the small interfering
RNAs (siRNAs). These RNAs do not arise from the hairpin structures
characteristic of miRNA precursors, but are instead processed from
long double-stranded RNA (dsRNA). Much of our understanding of
miRNA has been deduced from studies of siRNA, as there is
considerable overlap between the processing of these two types of
small RNAs. Thus, the differences between miRNA and siRNA are
informative to their role in immunity. In this review, we will compare
and contrast the processing of both these small RNAs subtypes.
The pathway of miRNA biogenesis is now well characterized
(reviewed by Cullen6). miRNAs originate from longer precursors
synthesized by RNA polymerase II with a stem-loop RNA structure
(Figure 1).7 After synthesis, these primary miRNAs (pri-miRNAs) are
cleaved at sites near the base of the stem by the nuclear RNase-III
nuclease, Drosha, to generate a 60–70 nt fragment named pre-miR-
NAs.8,9 The pre-miRNAs are then exported to the cytoplasm by
Exportin-5, and cleaved by a second RNase-III nuclease, Dicer, at
sites near the loop.10–12 The resulting miRNA is then integrated into
an miRNA ribonucleoprotein complex (miRNP). This contrasts with
siRNAs, which are generated from long dsRNAs. The source of the
dsRNA can be exogenous or endogenous via pairing of antisense RNA
to mRNA.13,14 As will be further discussed, translational silencing of
exogenous RNA by siRNA appears to be a major protective mechan-
ism against mobile genetic elements, such as transposons and invading
viruses in lower organisms. However, there are disparities in the
processing of siRNA and miRNA between lower and higher organ-
isms. These disparities have consequence to the function of these small
RNAs in the immune response in different species.
MECHANISM OF ACTION
The miRNP-silencing complex promotes transient mRNA transla-
tional repression via involvement of the Argonaute proteins (four in
mammals; Ago1, 2, 3 and 4).15–17 In contrast, siRNAs do not simply
repress translation but degrade its target mRNA that is complemen-
tary in sequence. Dicer processes long dsRNA into siRNAs of
approximately 20 nt.18 The siRNA is then incorporated into the
RNA-induced silencing complex (RISC) that then promotes specific
cleavage of homologous target mRNA at a single site in the middle of
the siRNA sequence.19 A few mismatches within the target mRNA
ablate the siRNA cleavage activity of RISC through Ago2 – the unique
Ago protein with cleavage activity.20,21 Unlike siRNAs, miRNAs bind
imperfectly to target mRNAs, almost universally in the 3¢ untranslated
region (3¢UTR). Target recognition is by pairing to a 7–8 nt miRNA
seed.22 To date, 450 miRNAs have been identified in humans, each one
possibly involved in the regulation of 100–200 target mRNAs.22–24
Nevertheless, miRNA targets are ill defined, as only 7–8 nt of com-
plementarity are required with the target for it to repress translation.22
In addition, multiple miRNAs can act cooperatively to repress mRNA
translation.25 miRNA and their target mRNAs accumulate in proces-
sing bodies (P-bodies) within the cytoplasm.26,27 Entry of an mRNA
Received 31 May 2007; accepted 6 June 2007
Centre for Cancer Research, Monash Institute of Medical Research, Monash University, Clayton, Victoria, Australia
Correspondence: Professor BRG Williams, Monash Institute of Medical Research, Monash Medical Centre, 246 Clayton Road, Clayton, Victoria 3168, Australia.
E-mail: Bryan.williams@med.monash.edu.au
Immunology and Cell Biology (2007) 0, 1–5
& 2007 Australasian Society for Immunology Inc. All rights reserved 0818-9641/07 $30.00
www.nature.com/icb

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into a P-body does not inevitably lead to its degradation, and miRNA-
dependent repression has been demonstrated to be reversible. In this
way, miRNA-repression can be regarded as repositories for untrans-
lated mRNA, allowing for fine-tuning of gene expression.
The above mechanism reflects the generally accepted process of
miRNA and siRNA post-transcriptional silencing. However, excep-
tions have been described. Instances have been reported where miRNA
also promotes RNA degradation through Ago2, as for siRNA, despite
the lack of perfect sequence complementarity.28,29 In addition, it has
been suggested that siRNAs have pretranscriptional effects by mod-
ulating histones and associated heterochromatin structure via an
RNA-induced transcriptional gene silencing-like complex in mam-
mals.30–32
THE LIMITATION OF RNA INTERFERENCE AS AN ANTIVIRAL
RESPONSE IN MAMMALS
The elucidation of the function of RNA interference (RNAi) in plants,
roundworm and fruit flies established that siRNA constitute a potent
antiviral pathway. The broad conservation of the essential components
of RNAi across species suggested a common function in higher
organisms. In mammals, however, the antiviral effects of RNAi remain
unsubstantiated.6 In accordance with observations in other species, the
use of transfected synthetic siRNA promotes cleavage of homologous
targets in mammalian cells.33 Nonetheless, detailed investigation of the
processes showed that siRNA uses the endogenous miRNA machinery.
The apparent lack of siRNA generation from viral dsRNAs in
mammalian cells was illustrated by Kobayashi et al.,34 who demon-
strated that the double-stranded reovirus could be inhibited by over-
expressing an siRNA (under the form of a short hairpin RNA
(shRNA)) specific to viral proteins. This revealed the lack of an
antiviral effect under normal conditions of infection. Moreover,
reovirus replication could be rescued by silent point mutations in
the viral sequences targeted by the shRNA.34 Accordingly, mammalian
Dicer appears incapable of processing long viral dsRNA into siRNAs.
These results are in accordance with the inability to detect siRNAs
during viral infections.6,35 In contrast, Dicer has been demonstrated to
process long hairpin RNA (lhRNA) into siRNAs and recruit target
silencing.36–44 Furthermore, it has been established that retrotranspo-
sons are processed into siRNAs in mammalian cells.45 Also, gene-
specific silencing can be induced by transfection of long
dsRNAs.41,46,47 This implies that Dicer has some capability to process
long dsRNA either transfected into or produced by the mammalian
cell. It is noteworthy that viral miRNAs have been found.35,48 This
implies that some viruses intentionally use the miRNA pathway to
infect the host.
RNAi AS AN ANTIVIRAL THERAPEUTIC
Although Dicer does not appear to be competent to generate siRNAs
from viral long dsRNA, RNAi can be used experimentally to prevent
viral replication (recently reviewed by Leonard and Schaffer,49 and
Morris and Rossi50). Both transfected siRNA and endogenously
expressed shRNA have potent antiviral activities.49 This underlines
the fact that siRNA/shRNA can enter the Ago2/miRNA machinery,
and recruit it to promote cleavage of viral RNAs. Several studies have

DICER
RIG-I
Figure 1 Regulation of innate immune sensing by miRNAs. (1) Sensing of pathogen-associated molecular patterns (PAMPs) via TLR or other intracellular
receptors (such as RIG-I) promotes signal transduction (2) and transcriptional induction of interferons and cytokines (3). Transcription of some pri-miRNAs
(such as miR-146a/b) is also induced. After Drosha processing the pre-miRNAs are recruited by Dicer, resulting in mature miRNAs. Association of miRNA-
Dicer with other proteins (such as Tar HIV-1 RNA-binding protein 2 – TRBP – Agoy) results in miRNP complex. Cytokine and interferon transcripts can
enter degradation/translation inhibition by miRNAs in P-bodies (4), or promote translation of protein (5).
miRNAs and innate immunity
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shown that the use of siRNA/shRNA-based antiviral strategies is
limited by viral evasion through rapid mutations of the target
sequences.44,51–53 To counteract this, different approaches have
employed a combination of several shRNAs,54 or endogenous expres-
sion of lhRNA.43,44 Although promising, these strategies should be
approached with care, given the possible saturation of endogenous
miRNA processing, as demonstrated with shRNAs.55 When over-
expressing lhRNAs in mammalian cells, we have detected activation
of nuclear factor-kB (NF-kB)56 and others have shown activation of
interferon-b (IFN-b),42 indicating that overexpression of some
lhRNAs can also instigate innate immune responses. This suggests
that better insights into the mechanisms of recognition of dsRNA in
mammalian cells are required before the use of RNAi as a therapeutic
antiviral in humans can be fully realized.
INTERPLAY BETWEEN RNAi COMPONENTS AND INNATE
IMMUNE PROTEINS
The observation that Dicer can process some endogenous and
transfected dsRNA begs the question why Dicer lacks direct antiviral
activity, but retains its processing ability in mammals. One explana-
tion may come from the observation that human Dicer activity is
impeded by extended RNA secondary structures with a base pairing
inferior to 100%.57 Accordingly, the processing of a hairpin structure
encoding for the miRNA, miR-122, was altered when it was cloned in
the context of the human hepatitis delta virus containing 74% base
pairing.57 Human Dicer has been shown to have a low level of
processing of dsRNA.58 The concept that different Dicer homologs
have different processing efficiencies is supported by the observation
that plants encode multiple Dicer homologs that are specialized to
respond to different types of virus.59 Another explanation for human
Dicer’s apparent inability to generate siRNA as an antiviral response
may lie in cooperative partners of the enzyme. Both mammals and C.
elegans only have one Dicer protein, yet these are involved in multiple
pathways.60 A major difference between mammals and nematodes is
the lack of an amplification machinery in mammals that relies on
RNA-dependent RNA polymerases (RdRPs). RdRPs amplify the 5¢-
region of the mRNA targeted by siRNA and turn initial single-
stranded RNA into dsRNA substrate for Dicer.61,62 Another significant
departure between mammals and lower organisms is the development
of an acquired immune system. It is speculated that much of the
innate immune response has become usurped into regulating acquired
immunity. In this paradigm, mammalian cells may have lost the RNAi
antiviral response, and instead rely on activation of pattern recogni-
tion receptors (PRRs) to detect exogenous dsRNA and elicit cytokine
and subsequent acquired immune responses. Examples of PRRs
include Toll-like receptor 3 (TLR3), retinoic acid inducible gene I
(RIG-I), melanoma differentiation-associated gene-5, 2¢-5¢ oligoade-
nylate synthetase and dsRNA-activated protein kinase (see Uematsu
and Akira63 for a recent review). Sensing of foreign cytoplasmic
dsRNA in mammals by RIG-I, for instance, has been shown to
correlate with the presence of 5¢-triphosphates of viral RNAs.64–66
RIG-I is homologous to C. elegans Dicer related helicase-1 (DRH-
1), essential for RNAi recruitment by long dsRNA.67,68 PIR1, an
additional partner of Dicer in C. elegans, is also conserved in
mammals.69 PIR1 is involved in the processing of the 5¢-triphosphate
groups generated by the RdRPs in C. elegans to generate better RNAi
substrates with a 5¢-monophosphate.69 The conservation of PIR1 in
the absence of mammalian RdRP, together with lack of detection of
viral-derived siRNA, suggests mammalian PIR1 activity has diverged
from RNAi to innate immunity. It is tempting to speculate that PIR1
could be recruited by RIG-I in the detection of 5¢-triphosphate dsRNA
in mammals. The divergence of the DRH-1/PIR-1/Dicer complex
from use with self-RdRPs amplification of RNAi in C. elegans, to a
putative RIG-I/PIR1-mediated sensing of foreign polyphosphated-
dsRNA in mammals, suggests a possible overlap between RNAi and
innate immunity that might have led to the appropriation of RNAi
constituents into cell signalling pathways.
A DIRECT ROLE FOR miRNA IN IMMUNITY
miRNAs are emerging as key regulators of many biological processes.
Much of the advancement of our understanding of cellular processes
modulated by miRNAs has focused on development. However, a role
for miRNAs in immune responses is gradually being uncovered
(Figure 1).
There is direct evidence of connectivity between the immune
response of mammals and miRNAs. The importance of miRNAs in
cell-mediated immunity is highlighted by Dicer conditional knockout,
which harbour T-cell deficiencies.70 Furthermore, both IFN-a and
IFN-aˆ have been shown to modulate the expression of several
miRNAs.71,72 It has also been discovered that the miRNA, miR-
146a/b, is upregulated in response to TLR2, 4 and 5 ligands in an
NF-kB-dependent manner.73 MiR-146a/b has been directly involved
in the translational regulation of tumour necrosis factor (TNF)
receptor-associated factor 6 and interleukin-1 receptor associated
kinase 1.73 This suggests a direct role for miRNAs as negative
regulators of innate immune activation in TLR signalling. These latter
studies relied upon micro-arrays containing miRNAs previously
identified outside of the context of an immune response.71–73 It is
envisioned that many miRNAs will only be induced during specific
immune responses and so strategies using the current miRNA micro-
arrays under-represent the extent of miRNAs induced during immune
responses. Therefore, an understanding of immunomodulatory
miRNA awaits a more comprehensive description of small RNAs.
Additional studies focusing on the post-transcriptional regulation
of mRNA show that the stability of IFN-regulated genes is regulated by
miRNAs via AU-rich elements (AREs) present in their 3¢UTR.74 AREs
had been established to shorten the half-lives of their mRNAs.74,75 It
has recently been shown that AREs are targeted for degradation by the
miRNA, miR-16, in mammalian cells.76 Accordingly, downregulation
of Dicer was accompanied by a loss of degradation of a reporter
containing the 3¢UTR of TNF-a. Also, inhibition of miR-16, using a
complementary oligonucleotide, reduced the degradation of similar
reporters.76 Tristetraprolin (TTP) was required for this miR-16-
mediated ARE-RNA decay.76 The p38 mitogen-activated protein
kinase (MAPK) pathway has previously been shown to regulate
ARE-containing mRNAs.77 Interestingly, this is also thought to
occur through phosphorylation of TTP.78–80 The finding that degra-
dation of some ARE-mRNAs can be mediated via the miRNP complex
in the decay centres with the help of TTP, adds to a growing picture of
finely tuned gene regulation.76 In this way, TTP may negatively
regulate some ARE-containing mRNA through miRNP/miR-16 degra-
dation under normal conditions, thereby limiting the expression of
inflammatory cytokines such as TNF-a. However, after expression of
additional stress signals by activation of the MAPK pathway, this
negative regulation of TTP/miR-16 may be alleviated via a phosphor-
ylation of TTP to stabilize ARE-mRNAs thus enhancing protein
expression.
CONCLUSION
The stimulation of PRRs activates signalling pathways, resulting in the
transcriptional induction of multiple genes. An additional level of
regulation is just beginning to be appreciated with the finding that
miRNAs and innate immunity
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miRNAs are able to fine-tune-induced genes at the protein level, via
regulation of mRNA half-life (via ARE-mediated degradation) or
inhibition of translation via cooperative binding in the 3¢UTR of
target genes. This regulation helps to balance the host immune
response and protect infected tissue. Further insights into the regula-
tion of cytokine signalling by miRNA should help design new
approaches to modulate inflammation in a clinical context.
ACKNOWLEDGEMENTS
Work in the authors’ laboratory is supported by NIH grants RO1 A134039 and
PO1 CA62220-11, and NHMRC Grant 436814.
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Immunology and Cell Biology