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r e v i e w 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 transcribed1, and the function of some of the resulting noncoding RNA is to regulate gene expression2. 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′ UTR4, 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 lncRNAGenome-wide analysis of lncRNA expression indicates that hundreds of lncRNAs are induced by inflammatory stimuli5,6 and that thousands are induced across the many stages of T cell development and activation7. In differentiated T lymphocytes, lineage-specific transcription factors are necessary for the expression of many lncRNAs7. 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 expression8. lncRNA can regulate transcription by directly binding transcription factors or participating in com-plexes that epigenetically control transcription (Fig. 2). The growth arrest–specific 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 arrest9. 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 transcription10. The NeST lncRNA encoded by the mouse virus-susceptibility locus Tmevp3 regulates viral load following infection with Theiler’s virus11. Located close to the gene encoding interferon-γ (IFN-γ), it is a constituent of a protein complex that regulates histone methylation and expression of the gene encoding IFN-γ (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 systemMartin 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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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 cells16. The efficiency and fidelity of polyadenylation is also linked to transcriptional regula-tion17 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 cells18.

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 expression19. Splicing is closely linked to the nonsense-mediated RNA decay (NMD) pathway, which removes

Post-transcriptional control begins in the nucleusIn the nucleus, capping of the RNA at its 5′ end, splicing, cleavage and polyadenylation are interlinked13. 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 RNAs14. 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 RNA15. The potential

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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-γ (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.

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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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transcripts with the potential to encode toxic truncated proteins20. NMD can also regulate gene expression directly in response to changes in splicing factor activity21. 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 expression22. During B cell activation, alternative polyadenylation can control the expression of protein-encoding genes independently of changes in mRNA abundance23.

The processing of noncoding RNA is also highly regulated, and the pathways of miRNA biogenesis are reviewed regularly24. RNA is also subject to many post-transcriptional covalent modifications15, includ-ing adenosine methylation, which has a role in regulating RNA stabil-ity25. RNA editing and the reversible addition of terminal nucleotides further diversifies the transcriptome26,27. The secondary structure of RNA is an intrinsic property and is probably subject to the influ-ence of RBPs or other RNAs28,29. It is affected by single-nucleotide polymorphisms (SNPs), including some that are associated with multiple sclerosis and asthma28. 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 nucleusMany 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 transcriptome30. 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 transduction31,32, 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 mRNA33. 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 contents35. 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 exosomes36. Various types of cells of the immune system can produce exosomes37–39, which may contain mRNA, miRNA and lncRNA40 ‘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 cells38. 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 RBPsA 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 2–7 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 AREs30. 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

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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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complexity in the recognition elements of such sequences41. 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 RBPs44,45. Redundancy is a recur-ring theme among RBPs with roles in the immune system, including regnase-1 (ref. 46) and the tristetraprolin (TTP) proteins47.

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-ples48 and provide routes of entry for manipulation49. 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-γ–activated inhibitor of translation’) complex50. 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-γ 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 response51. 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 eventsRBPs facilitate very rapid response to signal-transduction pathways, and the activity or participation of RBPs in multiprotein complexes can be regulated by phosphorylation52 or other forms of covalent modification (Fig. 4). The biogenesis of miRNAs, mediated by the multiprotein ‘microprocessor’ complex, is highly coordinated by envi-ronmental cues53. 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 precursors54. 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 activation55. 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 proteolysis56,57. More than a dozen RBPs contain the RING E3 ubiquitin ligase domain58, and the ligase activity of one of these (MEX3C) is necessary for it to mediate destabilization of HLA-A2 mRNA59. 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-nity60. 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)

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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 granules61. ADP-ribosylation of Ago2 attenuates its ability to promote miRNA-mediated repression61. 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 neutrophils62. 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 networksThe 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 partners63. 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 Arid5a65, or between RBPs and miRNAs, for bind-ing to mRNA can determine mRNA fate66. 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′ UTR67. The sequence that binds roquin-1 and roquin-2 is present in their mRNA42, and HuR regulates its own polyadenyla-tion68. Additionally, networks of splicing factors may regulate their own gene expression through NMD21. 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 RBPs69. 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 RBP70.

Similarities between the sequences of lncRNAs and those of the UTRs of protein-encoding genes may reflect conserved regulatory principles71,72. 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-actions75. 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 differentiation76. 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 CCR4–NOT 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 splicing78.

Noncoding RNA and RBPs regulate key immunological processesThe 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 RBPs30. 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 lineages79. 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 leukemia47. The ability of these RBPs to act as tumor suppressors is apparent in other systems, including human lymphoma80. 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. Roquin81, regnase-1 (ref. 82) and TTP67 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 inflammation83. TIA-1 is also an anti-inflammatory RBP in mouse lung models of inflammation84. 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 differentiation85. 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 thymus86,87, regulate the differentiation of follicular helper T cells88,89 or drive malignant transformation90. 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 pathway91. Key pathways involved in inflammation and cancer, such as that involving the transcription factor NF-κB, are regulated by multiple miRNAs, including miR17–92, 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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proteins that mediate shuttling of NFAT between the nucleus and cytoplasm92,93 (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 receptor–activated macrophages or TNF-treated fibroblasts express lncRNAs that promote and inhibit inflammatory gene expres-sion or act as feedback inhibitors of the NF-κB pathway5,6. 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 immunityRNA 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 expression94. 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 cell95.

Host-encoded RBPs also have direct roles as antiviral effectors. Some RBPs, including regnase-1, recognize and trigger the destruc-tion of viral RNA96,97. Zinc-finger antiviral protein (ZAP (PARP-13)) was discovered as a restriction factor for retroviruses98. 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 cells99, but not mouse cells100, 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 types101,102. 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 response103. Notably, virus-encoded miRNAs manipulate host-cell gene expres-sion104. Moreover, it is known that some viruses exploit host-cell miRNAs to their own advantage105,106; 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 miRNAs107.

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 mRNA108. A noncoding RNA produced by flavivirus inhibited the exonuclease XRN1 and thus affects the stability of host mRNA109. 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 HuR110. The Epstein-Barr virus genome encodes lncRNAs, such as Epstein-Barr virus–encoded small RNAs, that bind host RBPs111 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 cytomegalovirus113. 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 miR17–92 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 cluster114. 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-λ3 mRNA115. This may promote viral replication. Additionally, there is evidence for evolutionary selection for a SNP in the 3′ UTR of IFN-λ3 mRNA that prohibits miRNA binding. Interestingly, the SNP also renders the 3′ UTR of IFN-λ3 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-λ3 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 growth116. 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.

AcknowlEdGMEnTsWe 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 InTEREsTsThe 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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