484 VOLUME 15 NUMBER 6 JUNE 2014 NATURE IMMUNOLOGY

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POST-TRANSCRIPTIONAL AND POST-TRANSLATIONAL CONTROL OF IMMUNITY
Precise control of gene expression is a key feature of the immune sys-
tem. Transcripts and their protein products need to be produced and
maintained at concentrations that are tightly controlled in specific
places and over time. The majority of the genome is transcribed
1
, and
the function of some of the resulting noncoding RNA is to regulate
gene expression
2
. In this Review, we will consider three classes of non-
coding RNA: long noncoding RNA (lncRNA), arbitrarily classified as
RNA longer than 200 nucleotides (nt)
3
and with diverse mechanisms
for realizing regulatory potential; the well-characterized microRNA
(miRNA) subset of small 21- to 23-nt noncoding RNAs, for which roles
have emerged in diverse immunological processes; and the untrans-
lated regions (UTRs) of mRNA, which contain regulatory sequences.
The main function of miRNA in animals is the regulation of RNA
stability and translation, which will be the focus of this Review. The
protein-encoding capacity of the mammalian genome is approximately
3%, and a similar proportion accounts for the UTRs of mRNA, which
suggests extensive regulatory potential. Notably, increasing organismal
complexity correlates with lengthening of the 3 UTR
4
, emphasizing
the importance of this region. In many cases, as with some interleukins,
the length of the UTRs can exceed that of the coding region (Fig. 1).
Here we will review examples in which noncoding RNA controls gene
expression at the transcriptional or post-transcriptional level through
physical interaction with RNA-binding proteins (RBPs) or other non-
coding RNAs. We will illustrate that miRNAs and some RBPs regulate
mRNA stability and translation directly, according to different rules.
By doing so, they functionally complement each other. Together with
lncRNA, they establish gene networks that efficiently respond to extra-
cellular signaling. Finally, we will consider these molecular pathways
in host-pathogen interactions.
Transcriptional control by lncRNA
Genome-wide analysis of lncRNA expression indicates that hundreds of
lncRNAs are induced by inflammatory stimuli
5,6
and that thousands are
induced across the many stages of T cell development and activation
7
.
In differentiated T lymphocytes, lineage-specific transcription factors
are necessary for the expression of many lncRNAs
7
. Few lncRNAs
have been characterized functionally, but they seem to mediate diverse
mechanisms, often involving small RNAs and RBPs as regulatory fac-
tors, to control gene expression
8
. lncRNA can regulate transcription
by directly binding transcription factors or participating in com-
plexes that epigenetically control transcription (Fig. 2). The growth
arrestspecific lncRNA GAS-5 is linked to the effects of rapamycin
on T lymphocytes and well illustrates the pleiotropy and connectivity
of various aspects of post-transcriptional control. In optimal growth
conditions, GAS-5 RNA is degraded, but in growth-arrested cells this
does not occur and the transcript accumulates, which contributes to
growth arrest
9
. Functionally, GAS-5 RNA both is a precursor for a
small RNA and is able to act as a decoy for the glucocorticoid recep-
tor (GR). By competing with GR DNA-binding sequences, GAS-5
suppresses GR-regulated gene transcription
10
. The NeST lncRNA
encoded by the mouse virus-susceptibility locus Tmevp3 regulates
viral load following infection with Theilers virus
11
. Located close to
the gene encoding interferon- (IFN-), it is a constituent of a protein
complex that regulates histone methylation and expression of the gene
encoding IFN- (ref. 12). To assemble complexes at specific chroma-
tin regions and bring about changes in transcription, lncRNAs can
interact with protein, DNA or additional RNAs. lncRNAs also regulate
gene expression post-transcriptionally.
Laboratory of Lymphocyte Signalling and Development, The Babraham Institute,
Babraham Research Campus, Cambridge, UK. Correspondence should be
addressed to M.T. (martin.turner@babraham.ac.uk).
Received 30 January; accepted 1 April; published online 19 May 2014;
doi:10.1038/ni.2887
Noncoding RNA and its associated
proteins as regulatory elements of the
immune system
Martin Turner, Alison Galloway & Elena Vigorito
The rapid changes in gene expression that accompany developmental transitions, stress responses and proliferation are
controlled by signal-mediated coordination of transcriptional and post-transcriptional mechanisms. In recent years,
understanding of the mechanics of these processes and the contexts in which they are employed during hematopoiesis
and immune challenge has increased. An important aspect of this progress is recognition of the importance of RNA-binding
proteins and noncoding RNAs. These have roles in the development and function of the immune system and in pathogen life
cycles, and they represent an important aspect of intracellular immunity.
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NATURE IMMUNOLOGY VOLUME 15 NUMBER 6 JUNE 2014 485
for chromatin to have a regulatory role in post-transcriptional events
is suggested by the enrichment for histone marks within exons and
at points of polyadenylation in human CD4
+
T cells
16
. The efficiency
and fidelity of polyadenylation is also linked to transcriptional regula-
tion
17
as well as to the availability of factors that regulate the cleavage
of nascent transcripts. The production of transcripts encoding surface
or secreted immunoglobulin M is an example
of this form of regulation in B cells
18
.
Genome-wide analysis indicates that the
scale of alternative splicing is immense,
with the majority of transcripts encod-
ing alternative isoforms. Splicing is highly
regulated and represents an important
point at which signal-transduction path-
ways influence gene expression
19
. Splicing
is closely linked to the nonsense-mediated
RNA decay (NMD) pathway, which removes
Post-transcriptional control begins in the nucleus
In the nucleus, capping of the RNA at its 5 end, splicing, cleavage
and polyadenylation are interlinked
13
. Coordination of these
processes determines the final structure of the transcript in terms of
protein-encoding potential, the length and content of the UTRs and
the association of trans-acting factors before export from the nucleus.
Much of the potential of cytoplasmic RNA to respond to regulatory
factors is therefore programmed during the primordial events of
transcription (Fig. 3). The activities of RBPs and lncRNA are essen-
tial to this process. Studies using high-throughput RNA sequencing
are illuminating the dynamics of RNA biogenesis. Methods that take
account of the subcellular localization of RNA in lipopolysaccharide
(LPS)-stimulated macrophages derived from mouse bone marrow
have identified many intron-containing transcripts among chromatin-
associated RNAs
14
. This suggests that nascent transcripts may be
retained on chromatin for some time until processed. This delay may
present a platform for the epigenetic marking of RNA
15
. The potential
Transcription factors recruited to Ifng by NeST lncRNA
DNA
Tmevp3 Ifng
NeST lncRNA
GAS-5 lncRNA
GR
GR
GR active in dividing cells
a
NFAT
NES
NFAT
NLS
NRON lncRNA
Stimulation
b
c
Nucleus
Cytoplasm
Circular lncRNA
miRNA
STAU1
TINCR
lncRNA
AAAAAA
mRNA
d
lncRNA p21
AAAAAA
mRNA
HuR
miRNA
mRNA degrading enzymes
CMV lncRNA
miRNA degradation
NMD of
lncRNA
Translation
repression
Degradation
Sequestration
Transcription factors
Sequestration of GR in
growth-arrested cells
Figure 2 Diverse lncRNA mechanisms.
(a) lncRNA may interact with transcription
factors and affect their association with DNA.
The lncRNA NeST recruits transcription
factors to the gene encoding IFN- (Ifng) to
induce transcription, whereas GAS-5 lncRNA
sequesters the glucocorticoid receptor (GR) from
DNA. GAS-5 lncRNA undergoes NMD in dividing
cells but accumulates in growth-arrested cells.
(b) NRON lncRNA has a scaffolding role in
the NFAT complex and affects shuttling of the
complex between the nucleus and cytoplasm.
NES, nuclear export sequence; NLS, nuclear
localization sequence. (c) Various lncRNAs act
as sponges that divert miRNA from binding
their mRNA targets (left). In some cases,
lncRNA binding causes miRNA to be degraded
(right). CMV, cytomegalovirus. (d) lncRNAs
interact with mRNA. In the case of TINCR,
the target mRNA is stabilized by recruitment
of the RBP STAU1; in contrast, lncRNA-p21
inhibits translation of its target mRNA. Binding
of the RBP HuR to lncRNA-p21 inhibits
this activity by promoting miRNA-dependent
degradation of lncRNA-p21.
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
IL-8
IL-17A
IL-7
IL-11
IL-15
IL-33
IL-17D
IL-10
IL-20
IL-13
IL-12B
IL-1A
IL-25
IL-34
IL-3
IL-2
IL-22
IL-24
IL-26
IL-16
IL-5
IL-12A
IL-1B
IL-6
IL-18
IL-23A
IL-31
IL-17C
IL-19
IL-32
IL-27
IL-9
IL-4
IL-21
IL-17B
5 UTR CDS 3 UTR Figure 1 Contribution of untranslated and coding sequence to the
length of human interleukin mRNAs. The proportion of each transcript
taken up by 5 UTR (pink), coding DNA sequence (CDS; black) and
3 UTR (blue) is shown. Transcripts are ranked according to the
percentage of the mRNA taken up by coding sequence. Transcript
information was downloaded from the Ensembl project of genome
databases with BioMart software. For genes that encode more than
one transcript, only transcripts annotated with UTRs were considered,
and the transcript with the longest coding DNA sequence was selected
to represent that gene.
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486 VOLUME 15 NUMBER 6 JUNE 2014 NATURE IMMUNOLOGY
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transcripts with the potential to encode toxic truncated proteins
20
.
NMD can also regulate gene expression directly in response to changes
in splicing factor activity
21
. Regulation of the site of cleavage of the
nascent transcript followed by polyadenylation varies 3 UTR length
and can determine responsiveness of transcripts to RBPs and miRNA.
Changes in polyadenylation-site use accompany the activation of B
cells and T cells, with generalized shortening of 3 UTRs and the
consequent loss of miRNA binding promoting gene expression
22
.
During B cell activation, alternative polyadenylation can control the
expression of protein-encoding genes independently of changes in
mRNA abundance
23
.
The processing of noncoding RNA is also highly regulated, and the
pathways of miRNA biogenesis are reviewed regularly
24
. RNA is also
subject to many post-transcriptional covalent modifications
15
, includ-
ing adenosine methylation, which has a role in regulating RNA stabil-
ity
25
. RNA editing and the reversible addition of terminal nucleotides
further diversifies the transcriptome
26,27
. The secondary structure
of RNA is an intrinsic property and is probably subject to the influ-
ence of RBPs or other RNAs
28,29
. It is affected by single-nucleotide
polymorphisms (SNPs), including some that are associated with
multiple sclerosis and asthma
28
. The pathways of transcription and
post-transcriptional control are coupled to determine the sequence,
structure and fate of the mRNA (Fig. 3). Upon exit from the nucleus,
RNA is cloaked in proteins that regulate transcript stability, translation
and localization and that may respond to signaling pathways.
Post-transcriptional control beyond the nucleus
Many studies have investigated the role of changes in mRNA stability
in shaping the transcriptome of cells of the immune system. Each has
provided evidence of contributions of RNA stability to gene-specific
and system-wide regulation of the transcriptome
30
. When RNA is
short-lived, its abundance is more sensitive to changes in the tempo
of transcription. Thus, RNA decay acts in concert with transcrip-
tion to define the temporal kinetics of expression. Notably, unstable
transcripts are often the products of genes that encode regulators
of transcription and signal transduction
31,32
, which suggests that
such processes are most susceptible to coordinated changes in
transcription and post-transcriptional regulation. The majority of
cytokine-encoding transcripts contain one or more AU-rich elements
(AREs) in their 3 UTRs that mediate stability. The ARE motif is
known to interact with various RBPs, which can either stabilize or
destabilize mRNA
33
. Few experiments have attempted to investi-
gate cis-acting sequences within UTRs in vivo. Notable among these
is that removal of part of the Tnf 3 UTR, which contains an ARE,
has revealed an inhibitory role for the ARE in the biosynthesis of
tumor-necrosis factor (TNF)
34
.
RNA decay and translational control are regulated by interactions
among RNA, proteins and noncoding RNA, which can take place in
structures readily detectable by microscopy. These include stress granules,
which are defined by the presence of ribosomal subunits; transla-
tionally inactive mRNA; and processing bodies (P bodies), which
are sites of RNA decay. These structures are dynamic and frequently
juxtaposed, which allows exchange of their contents
35
. Furthermore,
the mobility of these structures in the cell offers the potential for
localized translation, the asymmetric segregation of cell contents
upon cytokinesis or the selective loading of cytoplasmic contents into
secreted exosomes
36
. Various types of cells of the immune system
can produce exosomes
3739
, which may contain mRNA, miRNA
and lncRNA
40
sampled in a nonrandom way from cytoplasmic
contents. Published studies have indicated the importance of RBPs
in the sorting of miRNA into the exosomes of activated T cells
38
.
The potential for exosomes to deliver selected cytosolic contents
between cells separated in time and space could represent an amenable
route for manipulation. Moreover, measurement of the noncoding
RNA content of exosomes in plasma may also prove useful as biomar-
ker providing information about biological processes taking place
in the host.
Comparison of the mechanisms of silencing by miRNAs and RBPs
A principal shared function of RBPs and miRNA in animals is the reg-
ulation of RNA stability and translation, mediated through physical
interaction with RNA. There is a considerable distinction between the
modes of RNA recognition used by miRNA and those used by RBPs.
The crucial miRNA-mRNA interaction takes place between the seed
region at nucleotide positions 27 within the 5 end of the miRNA, and
the miRNA-recognition elements of the target transcript (which often,
but not always, reside within the 3 UTR). These interactions promote
association with the multiprotein RNA-induced silencing complex
(RISC) and are based on primary structure (sequence) specificity.
Thus, they can potentially accommodate all possible base permuta-
tions and provide ample diversity in binding sites. Many RBPs interact
with short single-stranded sequences with limited complexity, such as
GU-rich elements, polypyrimidine tracts or AREs
30
. Each sequence
may interact with a variety of proteins with distinct RNA-binding
motifs and/or opposing functions. Several large-scale studies aimed
at defining sequences bound by RBPs have indicated limited
3 AAAAAAAAAAA
5
Exon 1 Exon 2 Exon 3
Cap binding complex
Coding sequence
DNA
5
Exon-intron boundaries
recognized and mRNA spliced
RBP
RBP
Exon 1
Intron
5 UTR 3 UTR
Exon 1 Exon 2 Exon 3 Intron Intron
Alternative splicing products
3 AAAAAAAAAAA
5
Exon 1 Exon 3
RBP
Long mRNA isoform
a
b
Short mRNA isoform
Co-transcriptional
processing and
association of
proteins and
complexes
Alternative polyadenylation sites
Exon junction
complexes
miRNA
binding site
PolyA binding
protein
Coding sequence
Figure 3 Processing in the nucleus regulates mRNA fate. mRNA
processing and association of RBPs occurs cotranscriptionally in the
nucleus. (a,b) Alternative splicing and alternative polyadenylation
affect the transcript structure, altering both the coding DNA sequence
encoded in the mRNA and the inclusion of regulatory sequences in the
UTRs. RBPs associate with the mRNA in the nucleus such that when
it exits into the cytoplasm it is already cloaked in regulatory factors.
(a) Example gene structure comprising three exons that can be spliced in
different ways to include or exclude exon 2. Two polyadenylation signals
are present, and the length of the 3 UTR depends on site use. Capping,
splicing, polyadenylation and RBP association occur cotranscriptionally.
(b) Two alternative mRNA isoforms; in this example, alternative splicing
gives rise to two coding sequences, and alternative polyadenylation leads
to the inclusion or exclusion of a miRNA and RBP-binding site.
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NATURE IMMUNOLOGY VOLUME 15 NUMBER 6 JUNE 2014 487
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complexity in the recognition elements of such
sequences
41
. RBPs also recognize secondary
structures of RNA. An example of this is the
constitutive decay element (CDE)
42
, a stem-
loop sequence that is bound by roquin-1 (an RBP identified in a genetic
screen for autoantibody-producing mutant mice)
43
and roquin-2.
Bioinformatics analysis has shown that this element is present in more
than 100 transcripts, including those encoding regulators of tolerance
and inflammation, in addition to those encoding molecules known
to be involved in the pathways of the function of follicular helper
T cells. This suggests that immunological regulation by roquin-1
and roquin-2 may involve a wide repertoire of RNA targets. Loss of
function of roquin-1 and roquin-2 in mature T cells has revealed
overlapping functions for these RBPs
44,45
. Redundancy is a recur-
ring theme among RBPs with roles in the immune system, including
regnase-1 (ref. 46) and the tristetraprolin (TTP) proteins
47
.
RNA secondary structures can be dynamic and determine whether
primary structures (sequences) are available for interacting with
miRNA or RBPs. Such dynamism may underpin regulatory princi-
ples
48
and provide routes of entry for manipulation
49
. Regulation of
growth factor VEGF-A translation in macrophages under hypoxia
provides a good example of this dynamic regulation. Hypoxia
induces exit from the nucleus of heterogeneous ribonucleoprotein
L (hnRNPL) and its subsequent binding to specific sequences in the
VEGF-A 3 UTR, which promotes mRNA stability and translation.
hnRNPL occludes binding of several miRNAs and also elicits a con-
formational change in the mRNA to promote release of the multi-
protein GAIT (IFN-activated inhibitor of translation) complex
50
.
It may seem surprising at first that one component of the GAIT
complex is the glycolytic enzyme GAPDH. However, moonlighting
functions for proteins as RBPs is an increasingly common finding.
Several studies have shown that GAPDH binds RNA and is a com-
ponent of complexes that regulate RNA. In T lymphocytes, GAPDH
is part of a complex with IFN- mRNA and may link the metabolic
program to the gene-expression program during the effector phase
Figure 4 RNA and RBP interact to regulate
gene expression. In this example of a signaling
network, noncoding RNA and an RBP work
in concert to regulate gene expression.
(a) A signal-transduction pathway leads to
post-translational modification (PTM) of RBPs,
which affects their activities in various ways.
(b) PTM of RBPs alters the localization of RBPs
and any associated RNAs to a cytoplasmic RNA
granule, such as a P-body or stress granule.
(c) PTM of RBPs affects the binding of other
proteins, which leads to the recruitment of
deadenylase by an RBP that destabilizes
its target mRNA. (d) PTM of RBPs affects
association with RNA, which leads to the
association of a stabilizing RBP with a lncRNA
that binds an mRNA. (e) PTM of RBPs affects
miRNA processing activity, which affects
the pool of miRNAs ready to bind mRNA
molecules. These miRNAs are more diverse
in sequence-binding ability than are RBPs.
(f) Multiple regulatory pathways converge
on an mRNA. RBPs and miRNA-RISC
complexes recruit common decapping and
deadenylation enzymes and may be inhibited
by a stabilizing RBP. Decay pathways also
affect translation initiation, which depends
on access to the 5 cap complex or internal
ribosomal entry sites (IRES), if present.
of the immune response
51
. It seems that functionally distinct RBPs
interact with a limited set of sequences and secondary structures to
bring about changes in mRNA fate. In contrast, miRNAs mediate
mainly silencing of mRNA through diverse sequence interactions.
Response to signaling events
RBPs facilitate very rapid response to signal-transduction pathways,
and the activity or participation of RBPs in multiprotein complexes
can be regulated by phosphorylation
52
or other forms of covalent
modification (Fig. 4). The biogenesis of miRNAs, mediated by the
multiprotein microprocessor complex, is highly coordinated by envi-
ronmental cues
53
. Modification of RBPs that are components of the
miRNA-processing machinery can lead to rapid global or specific
changes in miRNA abundance. The splicing and regulatory protein
KSRP associates with Drosha and Dicer and, in response to stimula-
tion of macrophages with LPS, enhances the processing of miR-155
precursors
54
. Proteolysis regulates RBPs either by removing them or
altering their functions. Proteolysis of Ago2, a catalytic component
of the RISC, underpins changes in miRNA associated with the RISC
during T cell activation
55
. The fact that several RBPs, including PTB
and HuR, are targets of caspase suggests modulation of RBP activity
during apoptosis. Upon activation of T cells or Toll-like receptors, the
RBP regnase-1 is cleared by proteolysis
56,57
. More than a dozen RBPs
contain the RING E3 ubiquitin ligase domain
58
, and the ligase activity
of one of these (MEX3C) is necessary for it to mediate destabilization
of HLA-A2 mRNA
59
. Roquin proteins also contain a RING domain,
and for roquin-2 this has been associated with the stress-induced ubiq-
uitination and degradation of ASK1, a kinase linked to innate immu-
nity
60
. Such findings raise questions about the extent to which the
degradation of RNA and that of protein are coordinated to bring about
changes in cellular function. Some RBPs contain poly(ADP-ribose)
3 AAAAAAAAAAA
3 AAAAAAAAAAA
3 AAAAAAAAAAA
3 AAAAAAAAAAA
3 AAAAAAAAAAA
3 AAAAAAAAAAA
3 AAAAAAAAAAA
3 AAAAAAAAAAA
5 cap
RBP
PABP
miRNA processing
pre-miRNA
Plasma membrane
Deadenylase
a
Activated cell surface receptor
RISC
RBP
b
c
d
Stabilizing
RBP
e
f
Ribosome
miRNA
processing
RBP
Mature miRNA
Diverse
miRNA pool
Decapping
enzyme
P body or
stress granule
Destabilizing
RBP
lncRNA
Signal
transduction
pathway
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488 VOLUME 15 NUMBER 6 JUNE 2014 NATURE IMMUNOLOGY
polymerase domains, which enables them to use NAD
+
to ribosylate
themselves or other granules, and glycohydrolases that degrade ADP-
ribose inhibit the assembly of stress granules proteins. This class of
proteins can promote the formation of stress granules, and glycohydro-
lases that degrade ADP-ribose inhibit the assembly of stress granules
61
.
ADP-ribosylation of Ago2 attenuates its ability to promote miRNA-
mediated repression
61
. The isomerization of serine or threonine-
proline bonds also regulates RBPs. The peptidyl-prolyl isomerase Pin1
associates with the ARE-binding protein hnRNPd (AUF1) and regu-
lates its interaction with mRNA encoding the cell-signaling molecule
GM-CSF in neutrophils
62
. After activation, hnRNPd bound to
GM-CSF mRNA is replaced with hnRNPc, which promotes stabiliza-
tion of the mRNA. Signal-mediated changes in miRNA abundance
can be regulated post-transcriptionally by RBPs. This notion extends
to lncRNAs, and it makes intuitive sense to speculate that RBPs can
control their stability. RBPs, such as HuR, that are involved in splicing,
polyadenylation, mRNA stability, translation and miRNA processing
are ideal targets for signal-mediated coordination of a particular
cellular program.
Noncoding RNAs and RBPs function in molecular networks
The propensity of miRNAs to be transcribed in polycistronic
operons sets them apart from the eukaryotic protein-encoding
genes and is suggestive of regulatory networks among targeted tran-
scripts. Transcriptome-wide analysis suggests such interactions are
extensive, involving hundreds of thousands of partners
63
. Similarly,
RBPs bind many functionally related transcripts, an observation
that has prompted comparison to operons, leading RBP networks
to been called regulons
64
. Competition between RBPs, such as
regnase-1 and Arid5a
65
, or between RBPs and miRNAs, for bind-
ing to mRNA can determine mRNA fate
66
. RBPs and miRNAs are
emerging as important regulatory hubs during dynamic changes in
gene expression. However, given the vast number of interactions
discovered, uncertainty remains about whether and how these
are consequential.
There is considerable evidence of autoregulation among RBPs; for
example, TTP regulates its own expression through binding to AREs
in its 3 UTR
67
. The sequence that binds roquin-1 and roquin-2 is
present in their mRNA
42
, and HuR regulates its own polyadenyla-
tion
68
. Additionally, networks of splicing factors may regulate their
own gene expression through NMD
21
. Analysis of evolutionarily con-
served sequences in 3 UTRs and data for RBP-mRNA interactions
suggests that RBPs such as HuR may be nodes for regulation among
multiple RBPs
69
. Networks among miRNAs and the mRNAs encoding
RBPs have not been clearly identified, although we predict that such
networks will be found. Such networks may include interactions of
miRNAs with RBPs, such as that described for hnRNPe2 and miR-
328, in which the miRNA acts as a competitive inhibitor to release
transcripts from regulation by the RBP
70
.
Similarities between the sequences of lncRNAs and those of the
UTRs of protein-encoding genes may reflect conserved regulatory
principles
71,72
. In fact, in addition to binding the 3 UTRs of mRNAs,
miRNAs can bind lncRNAs, genetically active pseudogenes and circular
RNAs (covalently linked RNAs that are generated by the head-to-tail
splicing of exons and show tissue-restricted expression)
73,74
. In
some instances, circular RNAs seem to function as miRNA sponges
decoys that release protein-encoding transcripts from miRNA sup-
pression (Fig. 2). Such observations have prompted a hypothesis of
competing endogenous RNAs that control mRNA-miRNA inter-
actions
75
. RBPs are probably also important for the function of this
regulatory network.
Additional layers of control are established by lncRNA-RBP
interactions and can promote either mRNA stabilization or mRNA
decay. The terminal differentiation-induced lncRNA TINCR interacts
with a wide range of mRNAs involved in epidermal differentiation
through a specific 25-nt sequence. Assisted by the RBP STAU1,
TINCR promotes transcript stabilization and cell differentiation
76
.
The lncRNA p21, in contrast, acts to inhibit translation of some
of its targets, a process that is inhibited by HuR, which promotes
miRNA-mediated degradation of p21 (ref. 77) (Fig. 2). Such exam-
ples exemplify the rich regulatory interactions of miRNAs, RBPs and
lncRNAs. Another point of convergence for miRNAs and RBPs is at
the core machinery for RNA degradation (Fig. 4). These include the
CCR4NOT deadenylase complex and the decapping enzymes DCP1
and DCP2. Although miRNAs may not engage NMD directly, they
exert indirect regulation of NMD through their control of key RBPs
that affect splicing
78
.
Noncoding RNA and RBPs regulate key immunological processes
The intertwined nature of miRNAs, lncRNAs and RBPs would suggest
their interaction in the control of a variety of cellular functions.
Numerous studies have linked miRNAs to the development and
function of lymphocytes, and evidence is emerging for roles for
RBPs
30
. Ectopic expression of the miRNA-binding protein Lin28B
in adult mouse hematopoietic stem cells (HSCs) reprograms them
to generate the fetal innate-like B cell and T cell lineages
79
. That is
probably mediated by effects on the let-7 family of miRNAs, whose
expression is inhibited by Lin28B. Importantly, the 3 UTR of Lin28B
mRNA is targeted by let-7 miRNA; however, the mechanism that
tips the balance of this negative feedback system remains to be dis-
covered. Many miRNAs act as tumor suppressors, and evidence of
this is also now extending to RBPs. Members of the TTP family limit
the developmental progression of early thymocytes and suppress the
development of T cell leukemia
47
. The ability of these RBPs to act as
tumor suppressors is apparent in other systems, including human
lymphoma
80
. Although RBPs have the potential to promote the
expression of genes encoding inflammatory molecules, the majority
of evidence so far suggests that they have an anti-inflammatory role.
Roquin
81
, regnase-1 (ref. 82) and TTP
67
can attenuate the inflam-
matory response, whereas HuR is often suggested to oppose TTP.
Despite that, HuR can act as a negative regulator of macrophage-
mediated inflammation
83
. TIA-1 is also an anti-inflammatory RBP
in mouse lung models of inflammation
84
. Thus, although many of
these RBPs regulate inflammatory and anti-inflammatory cytokines,
a common theme is that their absence tips the balance toward
inflammatory activation of the immune system.
miRNAs are linked to the regulation of transcriptional and
signal-transduction networks of lymphocyte development and T cell
differentiation
85
. miRNAs achieve this in part by regulating multiple
components of signal-transduction pathways to bring about an over-
all change in the activity of the pathway. Such interactions can set
the threshold for T cell antigen receptor selection in the thymus
86,87
,
regulate the differentiation of follicular helper T cells
88,89
or drive
malignant transformation
90
. A further example is miR-155, which
renders CD8
+
T cells resistant to the inhibitory effects of type I
interferons by targeting multiple components of the type I interferon
signaling pathway
91
. Key pathways involved in inflammation and
cancer, such as that involving the transcription factor NF-B, are
regulated by multiple miRNAs, including miR1792, miR-155 and
miR-146. Signaling pathways are also regulated by lncRNAs, such as
NRON, which inhibits the transcription factor NFAT. Rather than
targeting RNA, NRON inhibits NFAT by interacting with various
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NATURE IMMUNOLOGY VOLUME 15 NUMBER 6 JUNE 2014 489
proteins that mediate shuttling of NFAT between the nucleus and
cytoplasm
92,93
(Fig. 2). It is important to note that the cellular func-
tions ascribed to lncRNAs are diverse. Among these, Tmevp3 and
an additional lncRNA regulate the migration of T helper type 2 cells
into the lungs. Other studies have linked lncRNAs to inflammation.
Toll-like receptoractivated macrophages or TNF-treated fibroblasts
express lncRNAs that promote and inhibit inflammatory gene expres-
sion or act as feedback inhibitors of the NF-B pathway
5,6
. We antici-
pate that further interactions between RBPs and noncoding RNA will
emerge as key regulators of cell function in the immune system.
RBPs, noncoding RNA and intracellular immunity
RNA and DNA viruses exploit host-cell post-transcriptional mecha-
nisms to ensure efficient replication. At the same time, viruses must
avoid host responses geared toward targeting viral transcripts or
genomes. The challenges posed by RNA viruses and virus-derived
mRNA have fuelled the evolution of mechanisms to sense and censor
viral RNA. Indeed, the viral-host cell interaction has been suggested
to be a selective force that drives the complexity of mammalian gene
expression
94
. Stress granules and P bodies, sites at which RNA is
regulated in the cytoplasm, are subverted by viruses to promote their
own replication via co-opting or inhibition of the post-transcriptional
processes of the host cell
95
.
Host-encoded RBPs also have direct roles as antiviral effectors.
Some RBPs, including regnase-1, recognize and trigger the destruc-
tion of viral RNA
96,97
. Zinc-finger antiviral protein (ZAP (PARP-13))
was discovered as a restriction factor for retroviruses
98
. It binds
directly to viral RNA and recruits deadenylase and exonuclease
complexes, as well as the decapping enzymes. ZAP localizes to RNA
granules, which suggests that these are the site of its antiviral activity.
In human cells
99
, but not mouse cells
100
, a variant of ZAP associates
with the RNA helicase RIG-I promoting interferon production.
RNA-mediated interference, long thought to be the provenance of
plants and invertebrates, also seems to be functional in some mamma-
lian cell types
101,102
. Embryonic stem cells are able to generate small
interfering RNA derived from RNA viruses. Moreover, neonatal mice
infected with nodavirus lacking an RBP-encoding virulence factor
produce virus-derived small RNAs, which suggestd that the virulence
factor may inhibit RNA-mediated interference. More-differentiated
cells and adult tissues do not seem to retain this ability, however, and
instead rely on RNA-sensing proteins and the interferon response
103
.
Notably, virus-encoded miRNAs manipulate host-cell gene expres-
sion
104
. Moreover, it is known that some viruses exploit host-cell
miRNAs to their own advantage
105,106
; therefore, host shut-off of the
RISC may also limit viral replication or pathogenesis. Indeed, double-
stranded RNA has been shown to trigger ADP-ribosylation and
degradation of Ago2, a component of RISC. ZAP has been linked to
this, although the mechanism is not understood. Consequently, the
RISC is inhibited, and this has been associated with the enhance-
ment of otherwise cell-toxic aspects of the interferon response that
in uninfected cells are kept in check by miRNAs
107
.
Viruses regulate host-cell RBPs; for example, foot-and-mouth-
disease virus induces cleavage of RBPs, which contributes to the
suppression of the translation of host-cell mRNA
108
. A noncoding
RNA produced by flavivirus inhibited the exonuclease XRN1 and
thus affects the stability of host mRNA
109
. Interaction of cytoplasmic
HuR with Sindbis virus RNA is necessary for viral gene expression
and replication but also subverts host gene expression that is nor-
mally reliant upon HuR
110
. The Epstein-Barr virus genome encodes
lncRNAs, such as Epstein-Barr virusencoded small RNAs, that bind
host RBPs
111
as well as short noncoding RNAs. Virus-encoded RNA
has a role in targeting endogenous miRNAs; thus, Herpesvirus saimiri
expresses noncoding RNAs that bind to and promote the degradation
of miR-27 (ref. 112). miR-27 is also targeted by an RNA encoded
by mouse cytomegalovirus
113
. Such interactions contribute to viral
replication, but it remains unclear what targets and processes are regu-
lated by miR-27. Human cytomegalovirus promotes the transcription
of the host polycistronic transcript encoding the miR1792 cluster,
but the virus also produces an evolutionarily conserved noncoding
RNA from its own genome that promotes the degradation of a subset
of the miRNAs generated from the cluster
114
. The viral noncoding
RNA mediates the selective degradation by specific base pairing but is
not itself degraded (Fig. 2). Infection with hepatitis C virus induces
the (mis)expression of endogenous miRNAs that bind to the 3 UTR
and inhibits the expression of IFN-3 mRNA
115
. This may promote
viral replication. Additionally, there is evidence for evolutionary
selection for a SNP in the 3 UTR of IFN-3 mRNA that prohibits
miRNA binding. Interestingly, the SNP also renders the 3 UTR of
IFN-3 less susceptible to decay mediated by ARE-binding proteins;
whether these proteins are induced by hepatitis C virus or have a role
in regulating the gene encoding IFN-3 remains to be established.
RNA-based mechanisms of immunity are not limited to viral
infection. Remarkably, the transfer of miRNAs from red blood cells
into Plasmodium falciparum inhibits parasite growth
116
. Together
these findings suggest that the molecular details of host-pathogen inter-
action exemplify a rich layer of regulation at the post-transcriptional
level. Understanding these mechanisms may prompt new approaches
to interfering with pathogen replication.
Here we have presented selected examples to illustrate how
noncoding RNAs and RBPs interact in the immune system.
The dynamic interactions between distinct classes of noncoding
RNAs and their interacting proteins are of fundamental importance
and potentially regulate all aspects of immunity. Deepening our
understanding of these interactions may facilitate the invention of
new immunomodulators.
ACKNOWLEDGMENTS
We thank M. Linterman, P. Katsikis, R. Newman and L. Webb for advice and
comments on the manuscript. Supported by the Biotechnology and Biological
Sciences Research Council, the Medical Research Council (M.T. and E.V.) and
Leukaemia and Lymphoma Research (A.G.).
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Reprints and permissions information is available online at http://www.nature.com/
reprints/index.html.
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