Gene expression programs undergo constant regulation to quickly adjust to environmental stimuli that alter the physiological status of the cell, like cellular stress or infection. Gene expression is tightly regulated by multilayered regulatory elements acting in both cis and trans. Posttranscriptional regulation of the 3′ untranslated region (UTR) is a powerful regulatory process that determines the rate of protein translation from mRNA. Regulatory elements targeting the 3′ UTR include microRNAs, RNA-binding proteins, and long noncoding RNAs, which dramatically alter the immune response. We provide an overview of our current understanding of posttranscriptional regulation of immune gene expression. The focus of this review is on regulatory elements that target the 3′ UTR. We delineate how the synergistic or antagonistic interactions of posttranscriptional regulators determine gene expression levels and how dysregulation of 3′ UTR–mediated posttranscriptional control associates with human diseases.
The human genome contains ∼20,000–25,000 protein-coding genes, yet they make up only 1–2% of the genome. Recent data suggest that a large proportion of the genome is also transcribed into noncoding RNA that potentially regulate cellular processes (1, 2). Precise control of gene expression is vital for the host to maintain homeostasis, as well as to mount a rapid and effective immune response during infection. This becomes critical for genes associated with immune responses because they have to be induced rapidly in response to cellular stress, infection, and inflammatory stimuli, as well as be turned off quickly to limit undesirable immune-pathology caused by persistent immune activation. For such precise control, the cellular machinery has evolved regulators at several stages from transcription to translation, fine-tuning gene expression. These include structural and chemical modifications of chromosomal DNA, transcriptional regulation, posttranscriptional control of mRNA, varying translational efficiency, and protein turnover. These mechanisms, in concert, determine the spatiotemporal control of genes to elicit an optimal immune response.
mRNA is composed of a protein-coding region and 5′ and 3′ untranslated regions (UTRs). The 3′ UTR is variable in sequence and size; it spans between the stop codon and the poly(A) tail. Importantly, the 3′ UTR sequence harbors several regulatory motifs that determine mRNA turnover, stability, and localization; thus, it governs many aspects of posttranscriptional gene regulation. Over the past decade, it has become increasingly apparent that these regulatory motifs are critical in modulating immune responses (3–5).
Immune genes are particularly good models to study posttranscriptional gene regulation because an adequate, properly dosed immune response depends on their rapid, but transient, expression. The importance of posttranscriptional regulatory elements in these processes is evident from the evolution of multiple instability motifs and polymorphisms in the 3′ UTR of immune genes associated with pathogen pressure. ILs, IFNs, and chemokines encode mRNA-instability motifs, such as adenylate uridylate (AU)-rich elements (AREs), constitutive decay elements, and stem loops, which are targeted by specific RNA-binding proteins (RBPs) to destabilize the mRNA (6). These genes also harbor microRNA-recognition elements (MREs), which fine-tune immune responses through more sequence-specific binding to the 3′ UTR (7). Recent evidence suggests that motifs in RNA secondary and tertiary structures interact with posttranscriptional regulators to dictate the transcript stability of immune genes. Dysregulated gene expression resulting from polymorphisms in 3′ UTR sequences or their interacting regulatory proteins are associated with diseases, including cancer, infection, and autoimmune disorders (5, 8, 9). Thus, it is clear that the “language” of the 3′ UTR needs to be decoded to comprehend and potentially engineer ideal immune responses.
This review provides an integrated overview of the mechanisms and components that act in an additive, synergistic, and/or antagonistic manner through interaction with the 3′ UTR of mRNA, hence defining the “posttranscriptional regulome” of the 3′ UTR for control of immune gene expression and its implications for immune-mediated diseases.
Immune regulation by microRNAs
Major players of the posttranscriptional regulation are endogenously encoded microRNAs (miRNAs). These small (20–25 nt) noncoding, ssRNAs were first discovered in Caenorhabditis elegans in 1993 (10) and are distributed widely in eukaryotes. Since then, the potential regulatory role for this class of small RNAs has become increasingly appreciated (11). Canonical miRNA binding to target mRNA is determined by the “seed region” at the miRNA 5′ end (nt 2–7/8), which perfectly matches the MRE in the 3′ UTR of target mRNA (12). A more recent study also demonstrated a mechanism of noncanonical miRNA–mRNA interaction that does not require perfect base pairing within the seed region but depends instead on G-bulge sites within target mRNA (13).
During miRNA-dependent gene silencing, a polyprotein complex, the miRNA-induced silencing complex (miRISC), is recruited by an miRNA to target mRNA. Two distinct mechanisms for miRISC-mediated silencing have been documented. Initially, binding of the miRISC to target mRNA was thought only to interfere with translation and protein synthesis by inhibiting ribosome assembly, interfering with translational initiation factors, or by blocking translation postinitiation. However, subsequent studies identified a major contribution of miRISC to mRNA deadenylation and degradation (14, 15).
miRNAs are important regulators of immune responses and are involved in nearly all aspects of the immune system, ranging from immune cell ontogeny to innate and adaptive immunity against infections. Chen et al. (16) identified miR-181, miR-223, and miR-142 as modifiers of hematopoietic lineage differentiation. Furthermore, the crucial role of miRNAs in immune cell development was demonstrated: T cell lineage–specific deletion of Dicer, an essential enzyme for miRNA processing, results in impaired T cell development and a dysregulated CD4+ T cell cytokine signature (17, 18). Likewise, differentiation into B1 cells is controlled by miR-150, which is required to downregulate c-Myb expression (19).
Of several miRNAs key to modulating adaptive immune responses, miR-155 is one of the most prominent. SHIP1, a major regulator of the biology of various hematopoietic cells, is targeted by miR-155 through its 3′ UTR, with impacts on immune cell physiology, malignancies, and autoimmune disorders (20, 21). Bradley and colleagues (22) and Rajewsky and colleagues (23) also demonstrated that miR-155–deficient mice present impaired B and T cell immunity, caused by diminished activation of T cells through dendritic cells, impaired germinal center responses due to decreased TNF levels in germinal center B cells, and increased c-Maf expression skewing T cell differentiation toward a Th2 phenotype. miR-155 expression driven by Foxp3 is crucial for developing thymic regulatory T cells, because it limits SOCS1 protein expression and, thus, indirectly increases sensitivity to IL-2 signaling required for regulatory T cell expansion (24). Gracias and colleagues also found that miR-155 induced during primary CD8+ T cell activation renders the cells resistant to the antiproliferative effects of type I IFN, thus enabling establishment of effector memory (25). For a more comprehensive overview of literature on miR-155, its functions in immune cell biology, and implications for autoimmunity, please refer to these reviews (26, 27).
Various other studies reviewed by Baumjohann and Ansel (28) highlight specific mechanisms of miRNA-mediated regulation of CD4+ T cell differentiation and plasticity. miR-182, which is induced in CD4+ T cells after stimulation with IL-2 regulates Foxo1 to promote clonal expansion (29). A study by Li et al. (30) identified that miR-181a fine-tunes T cell sensitivity and selection during thymic development. Finally, miRNA targeting extends to effector cytokines, such as IFN-γ (IFNG), which harbors a conserved miR-29 MRE in its 3′ UTR. Several studies examined the role of miR-29 in regulating IFNG expression and identified miR-29 affecting IFNG mRNA stability directly or indirectly through targeting of TBET and EOMES mRNA (31, 32).
miRNAs are also important regulators of innate immune-sensing pathways, as initially shown by Baltimore and colleagues (33). They identified that miR-146 acts as a negative regulator of TLR4 signaling by targeting TLR adapters, TRAF6 and IRAK1, upon induction via its NF-κB–dependent promoter. Thus, miR-146 plays an important role in preventing excessive antimicrobial inflammatory responses. In line with these findings, miR-146a−/− mice develop spontaneous inflammation that progresses with age and leads to the development of myeloid malignancies (34, 35). TLR signaling is also regulated by the miRNAs let-7i, miR-145, miR-155, and miR-346, which target receptors or downstream adapter molecules; of these, miR-155 correlates directly, whereas let-7i correlates inversely, with TLR signaling and immune response (36, 37). Two other studies showed a role for miR-223 in granulocyte development and function: miR-223–deficient mice displayed increased granulocyte numbers, hypersensitivity to stimulation, and suppressed neutrophil activation (38, 39).
Sensing of pathogen-derived components by endosomal and cytosolic pattern recognition receptors induces innate immune responses. Expression of type I and type III IFNs is a hallmark of early innate immune response against viral infection. A variety of miRNAs regulate IFN-mediated immune responses by targeting IFN transcripts, the type I IFNR, and/or downstream transcription factors (5). We recently discovered that infection with hepatitis C virus induces expression of miR-208b and miR-499a-5p that target IFNL2 and IFNL3 genes (40). We also found that these miRNAs target IFNAR1 mRNA and, thus, control responses to type I IFN (A.P. Jarret and R. Savan, unpublished observations). Interestingly, although most miRNAs are endogenously encoded by the host genome, some viruses are known to encode their own viral miRNAs, which are predominantly involved in manipulation of the host immune responses (41, 42).
Because miRNAs fine-tune immune responses through control of immune gene expression, dysregulated miRNA expression has been linked to autoimmune diseases. One of the best-studied miRNAs in this context is miR-146, whose expression is decreased in systemic lupus erythematosus (SLE) patients, leading to elevated levels of type I IFN, a key characteristic of SLE (43). In contrast, rheumatoid arthritis (RA) patients present higher miR-146 expression, which, in turn, downregulates proinflammatory cytokines, such as TNF-α and IL-17 (44). Other miRNA signatures distinguish the two diseases: miR-155 and miR-15a are increased in SLE mouse models and affect regulatory T cell activity and production of anti-dsDNA Abs by B cells (44). miR-155 is also increased in RA patients alongside miR-132 and miR-16 (45). Ectopic expression of the miR-17-92 cluster in the lymphocyte compartment of mice results in lymphoproliferative disease and autoimmunity (46). A recent study showed that, in addition to miR-146, miR-155 is involved in the regulation of chronic inflammation (21). Several excellent reviews (5, 47–49) discuss the roles of miRNAs in immune regulation and immune responses in more depth.
Altogether, these examples demonstrate the role of miRNAs as critical modulators of the immune system, controlling expression of genes involved in immune cell ontogeny, innate and adaptive immune responses, as well as autoimmunity. Future investigations into posttranscriptional regulation through miRNAs will help to identify novel miRNA targets and mechanisms of immune regulation.
Immune regulation by RBPs
The 3′ UTRs harbor sequence or structural motifs that serve as recognition sites for RBPs that can affect mRNA stability. The best-characterized RBP recognition motif is the ARE, which is found in 8–10% of the human transcriptome. AREs can range from 40 to 150 nt in length and characteristically contain at least one AUUUA pentamer flanked by AU-rich sequence stretches (50–52). Almost three decades ago, the ARE motifs were first identified in human and mouse TNF genes (53). Shaw and Kamen (54) provided the first direct evidence that AREs influence mRNA stability by introducing an AU-rich sequence from the human GMCSF gene into the 3′ UTR of the rabbit β-globin gene, which resulted in drastic decay of β-globin mRNA. Several subsequent studies identified AREs in proto-oncogenes, transcription factors, IFNs, and cytokines, suggesting a major contribution of ARE-mediated decay to those genes (55).
Various motifs that facilitate interaction of RBPs with mRNAs have been identified, including, but not limited to, cytidylate uridylate–rich elements, guanylate uridylate–rich elements, repetitive C-rich sequences, and constitutive decay elements, all of which determine mRNA stability (6). Classically, RBP-mediated decay begins with binding of an RBP to its respective target mRNA, leading to recruitment of deadenylases to remove the poly(A) tail, followed by 3′ to 5′ exonucleolytic mRNA degradation, a process called ARE-mediated decay (AMD) when it occurs via an ARE motif. RBPs also were shown to mediate 5′ decapping and 5′-to-3′ mRNA degradation; conversely, they can also enhance mRNA stability by protecting it from other decay proteins.
One of the best-studied RBPs targeting AREs is tristetraprolin. Mouse and human tristetraprolin were first described in the early 1990s, as proteins encoded by the Zfp36/ZFP36 gene (56). Tristetraprolin and similar RBPs contain two tandem repeats of zinc finger motifs that enable direct interaction with AREs within the 3′ UTR of target mRNAs. Tristetraprolin was first shown to act by recruiting the exosome to ARE mRNAs to cause 3′-to-5′ exonucleolytic mRNA decay (57). However, Lykke-Andersen and Wagner (58) observed that tristetraprolin also interacts with enzymes involved in mRNA decapping and 5′-to-3′ exonuclease activity, suggesting additional mechanisms of tristetraprolin-mediated mRNA decay. More recently, another study demonstrated that tristetraprolin also interacts with the CCR4–CAF1–NOT deadenylation complex, possibly facilitating poly(A) tail deadenylation (59). The importance of tristetraprolin in gene regulation is evident, because Zfp36−/− mice develop a severe systemic inflammatory syndrome with patchy alopecia, dermatitis, erosive arthritis, cachexia, conjunctivitis, myeloid hyperplasia, glomerular mesangial thickening, and antinuclear Abs within 8 wk after birth (60). Posttranscriptional regulation by tristetraprolin is important in controlling the expression of various cytokine genes, especially inflammatory cytokines like TNF, GMCSF, IFNG, IL10, IL12, IL17, IL23, CCL3, and CXCL1 (61–70). Furthermore, linkage of a polymorphism in the ZFP36 gene with development of RA was reported (71). However, the genetic risk score for this polymorphism in RA is low, and this phenotype occurs only in a subset of analyzed individuals and appears to be specific to their ethnicity. The same class of zinc finger proteins includes the tristetraprolin paralogs butyrate response factors 1 and 2. Of these, butyrate response factor 1 was shown, using a functional cDNA library–cloning approach, to stabilize IL3, IL6, GMCSF, and TNF transcripts in an ARE-dependent manner (72, 73).
KH-type splicing regulatory protein (KSRP) is another RBP important for immune regulation, especially in controlling cytokine expression. Similar to tristetraprolin, KSRP participates in exosome-mediated mRNA decay of immune mediators like TNF, CXCL2, CXCL3, IL2, IL6, and IL8 (57, 74, 75). KSRP also controls Ifna4 and Ifnb expression, such that its absence increases resistance to viral infection due to rescue of IFN levels (76). In addition to its role in posttranscriptional control of immune genes, KSRP participates in miRNA maturation and processing (77) by forming a complex with Drosha and Dicer and interacting with the terminal loop of target precursor miRNA.
Three zinc-finger RBPs were recently identified that destabilize immune genes. Interaction of roquin-1 (RC3H1) with the 3′ UTR of Icos mRNA limits Icos expression in T cells (78, 79). Srivastava et al. (80) identified a novel role for roquin in miRNA homeostasis through direct interaction with Argonaute2, a central component of miRISC. They also showed that roquin controls levels of miR-146a, an miRNA targeting Icos mRNA, which may explain another way in which roquin modulates Icos. Mutation of Rc3h1 in sanroque mice results in an autoimmune disorder caused by accumulation of lymphocytes (81) and increased TNF-α levels (82). Rc3h2 has a redundant role in Icos mRNA downregulation (83, 84). Similar to roquin and tristetraprolin, regulatory RNase 1 (regnase-1)-deficient mice develop a systemic inflammatory phenotype that is caused by increased production of IL-6 and IL-12p40 (85). Furthermore, regnase-1 constitutively regulates expression of Rel, Tnfrsf4, and Il2 in naive T cells through 3′ UTR targeting and cleavage of mRNA (86). Upon triggering of the TCR, the Malt1 paracaspase cleaves regnase-1 to relieve suppression of its targeted genes and, thus, allow timely effector T cell activation and expansion (86, 87). Interestingly, although regnase-1 and roquin share a specific set of target mRNAs, a recent study demonstrated differential mRNA specificity between the two that depends on subcellular localization and translational status of the respective target mRNA (88).
The human Ag receptor (HuR) identified in 1996 (89) is a ubiquitously expressed member of the embryonic lethal abnormal vision–like protein family that harbors three RNA-binding domains that interact with AREs. In contrast to the destabilizing RBPs mentioned above, HuR overexpression stabilizes many ARE-containing mRNA transcripts (90, 91), including TNF (92, 93), suggesting competition between RBPs for mRNA binding and a dynamic interplay of posttranscriptional regulatory elements. For example, Ogilvie et al. (94) proposed a model of direct competition for IL2 mRNA binding by HuR and tristetraprolin. Binding of HuR to the mRNA prevents tristetraprolin-mediated recruitment of the exosome to the transcript. Furthermore, dysregulation of HuR–tristetraprolin equilibrium was shown to drive colon carcinogenesis, in which loss of tristetraprolin expression and simultaneous elevated expression of HuR greatly increase tumorigenic COX-2 expression (95). However, in recent years, it was found that HuR can also destabilize mRNA, mainly through concerted actions with miRISC (96). Thus, the effect of HuR on mRNA stability depends on the composite context of regulatory elements on the 3ʹ UTR. Likewise, AU-rich element-binding protein 1 (AUF1) can either stabilize or destabilize immune gene transcripts in a cell type–dependent manner that is not completely understood. AUF1 controls posttranscriptional regulation of numerous immune mediators like IL-3, IL-10, TNF-α, and GM-CSF (97–101), as well as the miRNA processing machinery by targeting and repressing DICER1 (102). A recent study established that AUF1 interacts with miRISC through Argonaute2 in either a cooperative or reciprocal manner, depending on which target mRNA is associated; this could explain, in part, the context-dependent differential effects of AUF1 (103).
Some RBPs were shown to interact with each other or compete for binding to the same recognition element, thereby having synergistic or antagonistic effects on stability of targeted mRNA (6). For instance, roquin and regnase-1 cooperate to regulate Th17 cell differentiation and expression of Th17 cell–promoting factors like IL-6 and ICOS (87). The dynamics of such RBP interactions and their effects on gene expression are determined by tissue- or cell type–specific expression of respective RBPs, as well as by external stimuli that alter the abundance or activity of certain RBPs. Notably, tristetraprolin levels and its extensive regulatory activity are altered by external stimuli. To start with, the p38 MAPK pathway is a critical regulator of tristetraprolin expression and activity (104, 105). MK2-mediated phosphorylation of tristetraprolin, which occurs after cytokine exposure and other stress-inducing external stimuli, leads to binding of 14-3-3 proteins, exclusion of tristetraprolin from stress granules, and, thereby, loss of tristetraprolin-dependent suppression of gene expression (106, 107). Fine-tuning of tristetraprolin activity through MK2-mediated phosphorylation stabilizes Tnf mRNA because it decreases tristetraprolin affinity to the ARE and its ability to compete with Tnf-stabilizing HuR for the same AREs (108).
The role of RBPs as immune regulators needs to be explored further to identify additional RBPs involved in immune regulation, delineate RBP mechanisms of mRNA degradation/stabilization, and identify novel RBP–mRNA interaction motifs.
Genetic variation within the 3′ UTR modulates posttranscriptional regulation in disease
Single-nucleotide polymorphisms (SNPs) of a gene are frequently associated with human diseases (40, 109–115). A single nucleotide change in the 3′ UTR can result in dysregulated posttranscriptional regulation. First, alterations in MRE sequences can change the affinity of the miRNA–mRNA interaction. Functional polymorphisms located directly within destabilization motifs have been reported for MREs. These SNPs disrupt the interaction sites for miRNA binding, which usually leads to stabilization of the mRNA transcript and increased protein levels. Such miRNA target site polymorphisms are linked to immune-associated diseases like cancer (114).
Genetic variation within the HLA-C gene associates with control of HIV infection (116) and HLA-C mRNA levels, as well as cell surface expression (117). We revealed a causal relationship between variations within the 3′ UTR of HLA-C mRNA and their effect on HIV control (118). Variation in the HLA-C 3′ UTR determines binding by miR-148a, directly affecting HLA-C expression and control of HIV. The SNP (rs10889677) in the IL23R 3′ UTR associates with inflammatory bowel disease, as it disrupts the MRE for let-7e and let-7f, resulting in increased IL-23R expression (119).
SNPs can also change the secondary and tertiary structure of the mRNA by changing base pair complementarity affecting stem-loop formation (120). Because 3′ UTR stem-loops act as scaffolds for RBP–mRNA interactions, such changes may disrupt interaction sites for RBPs and affect mRNA stability (121). We identified a functional SNP, rs4803217, within the 3′ UTR of IFNL3, which was tagged previously as one of the stronger predictors of natural and therapy-induced HCV clearance. This SNP dictates the turnover of IFNL3 mRNA by influencing the extent of AMD and miRNA-mediated decay (40). We identified the HCV-induced miRNAs dictating IFNL3 mRNA instability (see above) and that rs4803217 allows escape of miRNA-mediated decay to increase IFNL3 mRNA expression. However, how the 3′ UTR SNP influences IFNL3 AMD and which regulatory components are involved remain unclear.
Taken together, genetic variation within the 3′ UTR of immune genes is a strong determinant of immune response. Sequence variations can disrupt binding sites for miRNAs and/or RBPs, altering their ability to regulate transcripts (as summarized in Fig. 1). New sequencing technologies have advanced the investigations to understand these interactions and mechanisms behind the many disease polymorphisms in noncoding regions.
Polyadenylation and 3′ UTR shortening dictate mRNA function and turnover
Alternative polyadenylation (APA) presents a powerful mechanism to modulate the strength of 3′ UTR–mediated regulation. Several polyadenylation sites can be found in a single 3′ UTR in a majority of human genes, giving rise to different isoforms of a specific gene transcript (122, 123). APA alters the length of the 3′ UTR, which may affect mRNA stability and localization and/or translation of the isoforms (124). mRNAs with shorter 3′ UTRs are generally more stable than those with longer 3′ UTRs because they encode fewer regulatory elements, allowing them to escape regulation (125). Regulation of APA of an mRNA transcript thereby controls mRNA stability and protein levels.
APA was shown to be involved in B lymphocyte differentiation and the switch from membrane-bound to secreted IgM. B cell protein levels of the essential polyadenylation factor CSTF2 affect alternative processing of IgM mRNA through the differential usage of poly(A) sites (126). Low levels of CSTF2 in early-stage B cells promote cleavage at the distal IgM poly(A) site, leading to expression of membrane-bound IgM. During B cell maturation, increasing concentrations of CSTF2 skew cleavage site selection toward a more proximal intronic poly(A) site, resulting in expression of secreted IgM. A similar role for APA and involvement of CSTF2 also were shown in effector T cell maturation (127). Naive T cells express two mRNA isoforms of the transcription factor NFATc at low levels. However, after differentiation into effector T lymphocytes and a second exposure to Ags, T cells rapidly start to express high amounts of a third, shorter NFATc isoform. This change is caused by increased CSTF2 levels, which lead to usage of a proximal APA site in the NFATc mRNA.
APA can also precipitate profound immune dysfunction and pathology. Genetic variation within the IRF5 gene has been attributed to an increased risk for SLE development (112, 128). One of three SNPs identified by Graham et al. (129) affects IRF-5 protein levels and associates with high a risk for SLE. This SNP creates an APA signal, increasing the stability of IRF5 mRNA by truncating the 3′ UTR. Other genes such as TREX1, TLR7, and CD247 also contain variants in their 3′ UTRs that associate with SLE risk in screened patient cohorts (130–132). A mutation in the single poly(A) site of FOXP3 mRNA was associated with development of immune dysfunction, polyendocrinopathy, enteropathy, X-linked (110); a single A-to-G mutation in the hexameric polyadenylation signal results in impaired polyadenylation, failure to terminate transcription, and decreased FOXP3 expression. In the case of proto-oncogenes, 3′ UTR shortening through APA is associated with development of cancer (133).
Thus, APA presents an efficient and powerful mechanism by which mRNA stability can be transiently increased within a short time frame through shortening of its 3′ UTR. However, we still do not know how often APA is used to regulate gene expression in immune cells. New deep-sequencing technologies will determine the extent of APA in immune genes under steady-state and activated conditions.
Long noncoding RNAs are sinks for posttranscriptional regulators
Recently discovered long noncoding RNAs (lncRNAs) are generally classified as noncoding RNAs with a size > 200 nt. The role of lncRNAs as overarching modifiers of gene expression became more evident in the last decade as thousands of novel lncRNA transcripts were identified by improved sequencing technology (134). lncRNAs interact with mRNAs, miRNAs, and RBPs to modulate mRNA splicing, mRNA stability, and translation, thus adding complexity to the posttranscriptional regulatory mechanisms determining gene expression and protein output. lncRNAs compete with miRNAs for binding to target mRNA, thus masking miRNA binding sites and repressing miRISC-mediated mRNA decay (135). Similarly, lncRNAs also interfere with miRNA-mediated gene regulation by acting as sponges to sequester miRNAs (136). Furthermore, lncRNAs also were shown to alter mRNA stability by recruitment of RBPs to the 3′ UTR (137). Outstanding reviews discuss the contribution of lncRNAs to the overall posttranscriptional regulatory machinery in more detail (138, 139).
Posttranscriptional regulome: interaction between regulators of the posttranscriptional machinery dictates gene expression
Rather than considering each of the above-mentioned regulatory elements individually, we propose a more dynamic model of posttranscriptional gene regulation, including synergistic or antagonistic interaction of several of these regulators. Jing et al. (140) were the first to show that miRNAs and RBPs can mediate mRNA degradation in a cooperative manner. Although this study uncovered that tristetraprolin-mediated TNF mRNA decay requires miR-16 processing by Dicer, as well as the presence of Argonaute, direct binding of miR-16 to tristetraprolin was not found. The investigators suggested an indirect interaction of miR-16 with tristetraprolin through association and complex formation with Argonaute family members (140). Later, miR-221 was found to associate with tristetraprolin to facilitate TNF mRNA decay (141). Furthermore, we showed that IFNL3 mRNA stability is determined by cooperative actions of miRNAs and RBPs targeting AREs in the IFNL3 3′ UTR (40). In contrast, miRNAs can also directly compete with RBPs for 3′ UTR binding. For example, the seed region of miR-466l is complementary to the pentameric AUUUA sequence of the ARE. In the presence of miR-4661, IL10 mRNA and protein increase (142), because miR-466l competes with tristetraprolin for the AUUUA motif and prevents tristetraprolin-mediated degradation. miR-29 also was identified as a stabilizer of tumor suppressor A20 mRNA through interaction with other posttranscriptional elements, where it acts as a RNA decoy for the RBP HuR, thus preventing A20 mRNA decay (143). Additionally, we uncovered a similar mechanism by which miR-29 stabilizes gene expression of IFNG through competitive binding to RBP-recognition elements. To study the simultaneous effects of miR-29 and AREs on IFNG mRNA stability, we retained RNA structural integrity by using constructs containing the complete 3′ UTR sequence. We observed that miR-29 stabilizes IFNG expression in the presence of the tristetraprolin complex (R. Savan and H.A. Young, unpublished observations). The AREs targeted by tristetraprolin for mRNA decay are in close proximity to the miR-29 binding site in the secondary mRNA structure. We propose that this prevents the recruitment of GW182 so that miRISC cannot degrade the transcript. Although miR-29 was shown to enhance degradation of IFNG (32), these studies used partial UTR sequences lacking the AREs and, thus, may have overlooked crucial interactions between miR-29 and tristetraprolin. Thus, overall, miR-29 acts as a stabilizer of IFNG mRNA through its antagonism of AMD.
Another prominent example of interaction between miRNAs and RBPs in gene expression control is the Lin28/let-7 axis and its role in hematopoiesis and cancer. The RBPs Lin28a and Lin28b bind to pre–let-7 to inhibit its processing by Drosha and Dicer (144). Lin28a/b also facilitate oligo-uridylation of pre–let-7 by TUT4, leading further to its decay (145). Yuan et al. (146) highlighted the importance of Lin28/let-7 balance in the developing immune system, when they found that Lin28b was expressed in fetal, but not in adult, lymphocyte progenitor cells and that Lin28b in these cells correlated inversely with expression levels of let-7 family members. Ectopic expression of Lin28 in bone marrow hematopoietic stem/progenitor cells represses let-7 miRNAs and allows multilineage reconstitution.
Many examples given in previous sections of this review combine diverse simultaneous posttranscriptional regulatory elements, which can either compete or cooperate. Given this ample evidence that the interplay of multiple factors can result in a vastly different phenotype from the actions of individual components, integrating these regulatory pathways while studying these regulations will provide a complete picture of the 3′ UTR “regulome” of the gene.
Posttranscriptional regulation through the 3′ UTR is a potent mechanism to control the development and homeostasis of the immune system and to quickly adjust the expression of immune genes upon stimulation by cell-intrinsic or cell-extrinsic cues. In this review, we discussed different mechanisms by which posttranscriptional regulation through the 3′ UTR of mRNA transcripts occurs. miRISC and AMD are the most powerful regulatory elements dictating mRNA stability and/or degradation. However, alteration of 3′ UTR sequence, structure, and length by SNPs and APA add an additional level of complexity to the actions of miRISC and AMD. Furthermore, lncRNAs interact directly with mRNA, miRNA, and RBPs to modify their individual activity as an additional layer of control (summarized in Fig. 1).
Not surprisingly, a number of immune-mediated diseases are associated with genetic variation within 3′ UTR elements of immune genes (Fig. 2). Identification of novel SNPs within the 3′ UTR allows deeper insights into the linkage between posttranscriptional dysregulation and human disease. Such variations can affect mRNA localization, stability, and translation efficiency, all of which ultimately dictate expression of a given gene. Future investigations should be aimed at the identification of novel posttranscriptional regulatory elements and their specific mode(s) of action, as well as a detailed understanding of how multiple elements act in synergy or in antagonism. Deep-sequencing and mass spectrometry approaches will help to uncover novel RNA–RBP interactions and provide mechanistic explanations for genetic data. Furthermore, RNA sequencing–based approaches will unravel novel miRNAs and lncRNAs and their possible target sites within specific 3′ UTRs. A recently published study by Zhao et al. (147) presents an excellent example of how novel sequencing approaches massively improve identification of novel cis-acting regulatory elements in the 3′ UTR and how genetic variation within these elements affects mRNA stability.
A better understanding of the complex interplay of regulatory elements interacting with the 3′ UTR will help to link immune disorders to dysregulation of posttranscriptional mechanisms. In addition, it may pave the way for development of novel therapeutic approaches and the design of genotype-specific, tailored treatment for immune diseases.
We thank Abigail P. Jarret, Chrissie Lim, Adelle P. McFarland, Snehal S. Ozarkar, and Justin A. Roby for critical reading of the manuscript. The authors apologize to all whose work could not be cited because of space limitations.
This work was supported in part by National Institutes of Health Grant 1R01AI108765 (to R.S.).
Abbreviations used in this article:
human Ag receptor
long noncoding RNA
miRNA-induced silencing complex
systemic lupus erythematosus
The authors have no financial conflicts of interest.