Translating the Untranslated Region Johannes Schwerk and Ram Savan J Immunol 2015; 195:2963-2971; ; This information is current as doi: 10.4049/jimmunol.1500756 of September 27, 2021. http://www.jimmunol.org/content/195/7/2963 Downloaded from References This article cites 147 articles, 51 of which you can access for free at: http://www.jimmunol.org/content/195/7/2963.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Th eJournal of Brief Reviews Immunology

Translating the Untranslated Region Johannes Schwerk and Ram Savan expression programs undergo constant regulation sequence and size; it spans between the stop codon and the to quickly adjust to environmental stimuli that alter the poly(A) tail. Importantly, the 39 UTR sequence harbors sev- physiological status of the cell, like cellular stress or in- eral regulatory motifs that determine mRNA turnover, stability, fection. is tightly regulated by mul- and localization; thus, it governs many aspects of posttranscrip- tilayered regulatory elements acting in both cis and tional gene regulation. Over the past decade, it has become in- trans. Posttranscriptional regulation of the 39 untrans- creasingly apparent that these regulatory motifs are critical in lated region (UTR) is a powerful regulatory process that modulating immune responses (3–5). determines the rate of translation from mRNA. Immune are particularly good models to study post- transcriptional gene regulation because an adequate, properly

Regulatory elements targeting the 39 UTR include Downloaded from , RNA-binding , and long non- dosed immune response depends on their rapid, but transient, coding RNAs, which dramatically alter the immune expression. The importance of posttranscriptional regulatory ele- ments in these processes is evident from the evolution of mul- response. We provide an overview of our current under- tiple instability motifs and polymorphisms in the 39 UTR of standing of posttranscriptional regulation of immune immune genes associated with pathogen pressure. ILs, IFNs, gene expression. The focus of this review is on regulatory and chemokines encode mRNA-instability motifs, such as elements that target the 39 UTR. We delineate how the adenylate uridylate (AU)-rich elements (AREs), constitutive http://www.jimmunol.org/ synergistic or antagonistic interactions of posttranscrip- decay elements, and stem loops, which are targeted by spe- tional regulators determine gene expression levels and cific RNA-binding proteins (RBPs) to destabilize the mRNA how dysregulation of 39 UTR–mediated posttranscrip- (6). These genes also harbor microRNA-recognition ele- tional control associates with human diseases. The ments (MREs), which fine-tune immune responses through Journal of Immunology, 2015, 195: 2963–2971. more sequence-specific binding to the 39 UTR (7). Recent evidence suggests that motifs in RNA secondary and tertiary structures interact with posttranscriptional regulators to dic- he contains ∼20,000–25,000 protein- tate the transcript stability of immune genes. Dysregulated by guest on September 27, 2021 coding genes, yet they make up only 1–2% of the ge- gene expression resulting from polymorphisms in 39 UTR nome. Recent data suggest that a large proportion of the T sequences or their interacting regulatory proteins are associ- genome is also transcribed into noncoding RNA that potentially ated with diseases, including cancer, infection, and autoim- regulate cellular processes (1, 2). Precise control of gene expres- mune disorders (5, 8, 9). Thus, it is clear that the “language” sion is vital for the host to maintain homeostasis, as well as to 9 mount a rapid and effective immune response during infection. of the 3 UTR needs to be decoded to comprehend and This becomes critical for genes associated with immune responses potentially engineer ideal immune responses. This review provides an integrated overview of the mecha- because they have to be induced rapidly in response to cellular nisms and components that act in an additive, synergistic, and/or stress, infection, and inflammatory stimuli, as well as be turned antagonistic manner through interaction with the 39 UTR of off quickly to limit undesirable immune-pathology caused mRNA, hence defining the “posttranscriptional regulome” of by persistent immune activation. For such precise control, the the 39 UTR for control of immune gene expression and its cellular machinery has evolved regulators at several stages from implications for immune-mediated diseases. transcription to translation, fine-tuning gene expression. These include structural and chemical modifications of chromo- somal DNA, transcriptional regulation, posttranscriptional con- Immune regulation by microRNAs trol of mRNA, varying translational efficiency, and protein Major players of the posttranscriptional regulation are endo- turnover. These mechanisms, in concert, determine the spatio- genously encoded microRNAs (miRNAs). These small (20–25 temporal control of genes to elicit an optimal immune response. nt) noncoding, ssRNAs were first discovered in Caeno- mRNA is composed of a protein-coding region and 59 and rhabditis elegans in 1993 (10) and are distributed widely in 39 untranslated regions (UTRs). The 39 UTR is variable in eukaryotes. Since then, the potential regulatory role for this

Department of Immunology, University of Washington, Seattle, WA 98109 Abbreviations used in this article: AMD, ARE-mediated decay; APA, alternative polyadenylation; ARE, AU-rich element; AU, adenylate uridylate; HuR, human Ag Received for publication April 1, 2015. Accepted for publication July 30, 2015. receptor; lncRNA, long noncoding RNA; miRNA, microRNA; miRISC, miRNA- This work was supported in part by National Institutes of Health Grant 1R01AI108765 induced silencing complex; MRE, microRNA-recognition element; RA, rheumatoid (to R.S.). arthritis; RBP, RNA-binding protein; SLE, systemic lupus erythematosus; SNP, single-nucleotide polymorphism; UTR, untranslated region. Address correspondence and reprint requests to Dr. Ram Savan, Department of Immunology, University of Washington, Seattle, WA 98109. E-mail address: Ó [email protected] Copyright 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500756 2964 BRIEF REVIEWS: TRANSLATING THE UNTRANSLATED REGION class of small RNAs has become increasingly appreciated sensitivity and selection during thymic development. Finally, (11). Canonical miRNA binding to target mRNA is de- miRNA targeting extends to effector cytokines, such as IFN-g termined by the “seed region” at the miRNA 59 end (nt 2– (IFNG), which harbors a conserved miR-29 MRE in its 7/8), which perfectly matches the MRE in the 39 UTR of 39 UTR. Several studies examined the role of miR-29 in regu- target mRNA (12). A more recent study also demonstrated lating IFNG expression and identified miR-29 affecting IFNG a mechanism of noncanonical miRNA–mRNA interaction mRNA stability directly or indirectly through targeting of that does not require perfect base pairing within the seed TBET and EOMES mRNA (31, 32). region but depends instead on G-bulge sites within target miRNAs are also important regulators of innate immune- mRNA (13). sensing pathways, as initially shown by Baltimore and col- During miRNA-dependent gene silencing, a polyprotein leagues (33). They identified that miR-146 acts as a negative complex, the miRNA-induced silencing complex (miRISC), regulator of TLR4 signaling by targeting TLR adapters, is recruited by an miRNA to target mRNA. Two distinct TRAF6 and IRAK1, upon induction via its NF-kB–dependent mechanisms for miRISC-mediated silencing have been docu- promoter. Thus, miR-146 plays an important role in prevent- mented. Initially, binding of the miRISC to target mRNA was ing excessive antimicrobial inflammatory responses. In line 2 2 thought only to interfere with translation and protein synthesis with these findings, miR-146a / mice develop spontaneous by inhibiting ribosome assembly, interfering with translational inflammation that progresses with age and leads to the de- initiation factors, or by blocking translation postinitiation. velopment of myeloid malignancies (34, 35). TLR signaling is

However, subsequent studies identified a major contribu- also regulated by the miRNAs let-7i, miR-145, miR-155, and Downloaded from tion of miRISC to mRNA deadenylation and degradation miR-346, which target receptors or downstream adapter mole- (14, 15). cules; of these, miR-155 correlates directly, whereas let-7i cor- miRNAs are important regulators of immune responses relates inversely, with TLR signaling and immune response and are involved in nearly all aspects of the immune system, (36, 37). Two other studies showed a role for miR-223 in ranging from immune cell ontogeny to innate and adap- granulocyte development and function: miR-223–deficient mice tive immunity against infections. Chen et al. (16) identified displayed increased granulocyte numbers, hypersensitivity to http://www.jimmunol.org/ miR-181, miR-223, and miR-142 as modifiers of hematopoi- stimulation, and suppressed neutrophil activation (38, 39). etic lineage differentiation. Furthermore, the crucial role of Sensing of pathogen-derived components by endosomal and miRNAs in immune cell development was demonstrated: T cell cytosolic pattern recognition receptors induces innate immune lineage–specific deletion of Dicer, an essential enzyme for responses. Expression of type I and type III IFNs is a hallmark miRNA processing, results in impaired T cell development of early innate immune response against viral infection. A variety and a dysregulated CD4+ T cell cytokine signature (17, 18). of miRNAs regulate IFN-mediated immune responses by tar- Likewise, differentiation into B1 cells is controlled by miR-150, geting IFN transcripts, the type I IFNR, and/or downstream

which is required to downregulate c-Myb expression (19). transcription factors (5). We recently discovered that infection by guest on September 27, 2021 Of several miRNAs key to modulating adaptive immune with hepatitis C virus induces expression of miR-208b and responses, miR-155 is one of the most prominent. SHIP1, a miR-499a-5p that target IFNL2 and IFNL3 genes (40). We major regulator of the biology of various hematopoietic cells, is also found that these miRNAs target IFNAR1 mRNA and, targeted by miR-155 through its 39 UTR, with impacts on thus, control responses to type I IFN (A.P. Jarret and R. Savan, immune cell physiology, malignancies, and autoimmune unpublished observations). Interestingly, although most miRNAs disorders (20, 21). Bradley and colleagues (22) and Rajewsky are endogenously encoded by the host genome, some viruses and colleagues (23) also demonstrated that miR-155–deficient are known to encode their own viral miRNAs, which are mice present impaired B and T cell immunity, caused by di- predominantly involved in manipulation of the host immune minished activation of T cells through dendritic cells, impaired responses (41, 42). germinal center responses due to decreased TNF levels in Because miRNAs fine-tune immune responses through con- germinal center B cells, and increased c-Maf expression skewing trol of immune gene expression, dysregulated miRNA expression T cell differentiation toward a Th2 phenotype. miR-155 ex- has been linked to autoimmune diseases. One of the best-studied pression driven by Foxp3 is crucial for developing thymic reg- miRNAs in this context is miR-146, whose expression is de- ulatory T cells, because it limits SOCS1 protein expression creased in systemic lupus erythematosus (SLE) patients, leading and, thus, indirectly increases sensitivity to IL-2 signaling to elevated levels of type I IFN, a key characteristic of SLE (43). required for regulatory T cell expansion (24). Gracias and In contrast, rheumatoid arthritis (RA) patients present higher colleagues also found that miR-155 induced during primary miR-146 expression, which, in turn, downregulates proin- CD8+ T cell activation renders the cells resistant to the anti- flammatory cytokines, such as TNF-a and IL-17 (44). Other proliferative effects of type I IFN, thus enabling establishment miRNA signatures distinguish the two diseases: miR-155 and of effector memory (25). For a more comprehensive overview miR-15a are increased in SLE mouse models and affect regu- of literature on miR-155, its functions in immune cell biol- latory T cell activity and production of anti-dsDNA Abs by ogy, and implications for autoimmunity, please refer to these B cells (44). miR-155 is also increased in RA patients alongside reviews (26, 27). miR-132 and miR-16 (45). Ectopic expression of the miR-17- Various other studies reviewed by Baumjohann and Ansel 92 cluster in the lymphocyte compartment of mice results in (28) highlight specific mechanisms of miRNA-mediated regu- lymphoproliferative disease and autoimmunity (46). A recent lation of CD4+ T cell differentiation and plasticity. miR-182, study showed that, in addition to miR-146, miR-155 is in- which is induced in CD4+ T cells after stimulation with IL-2 volved in the regulation of chronic inflammation (21). Several regulates Foxo1 to promote clonal expansion (29). A study excellent reviews (5, 47–49) discuss the roles of miRNAs in by Li et al. (30) identified that miR-181a fine-tunes T cell immune regulation and immune responses in more depth. The Journal of Immunology 2965

Altogether, these examples demonstrate the role of miRNAs IL10, IL12, IL17, IL23, CCL3,andCXCL1 (61–70). Further- as critical modulators of the immune system, controlling more, linkage of a polymorphism in the ZFP36 gene with de- expression of genes involved in immune cell ontogeny, innate velopment of RA was reported (71). However, the genetic risk and adaptive immune responses, as well as autoimmunity. score for this polymorphism in RA is low, and this phenotype Future investigations into posttranscriptional regulation through occurs only in a subset of analyzed individuals and appears to be miRNAs will help to identify novel miRNA targets and mech- specific to their ethnicity. The same class of zinc finger proteins anisms of immune regulation. includes the tristetraprolin paralogs butyrate response factors 1 and 2. Of these, butyrate response factor 1 was shown, using Immune regulation by RBPs a functional cDNA library–cloning approach, to stabilize IL3, The 39 UTRs harbor sequence or structural motifs that serve IL6, GMCSF,andTNF transcripts in an ARE-dependent as recognition sites for RBPs that can affect mRNA stability. manner (72, 73). The best-characterized RBP recognition motif is the ARE, KH-type splicing regulatory protein (KSRP) is another RBP which is found in 8–10% of the human transcriptome. AREs important for immune regulation, especially in controlling can range from 40 to 150 nt in length and characteristically cytokine expression. Similar to tristetraprolin, KSRP participates contain at least one AUUUA pentamer flanked by AU-rich in exosome-mediated mRNA decay of immune mediators sequence stretches (50–52). Almost three decades ago, the like TNF, CXCL2, CXCL3, IL2, IL6,andIL8 (57, 74, 75). ARE motifs were first identified in human and mouse TNF KSRP also controls Ifna4 and Ifnb expression, such that its genes (53). Shaw and Kamen (54) provided the first direct absence increases resistance to viral infection due to rescue of Downloaded from evidence that AREs influence mRNA stability by introducing IFN levels (76). In addition to its role in posttranscriptional an AU-rich sequence from the human GMCSF gene into the control of immune genes, KSRP participates in miRNA mat- 39 UTR of the rabbit b-globin gene, which resulted in drastic uration and processing (77) by forming a complex with decay of b-globin mRNA. Several subsequent studies iden- Drosha and Dicer and interacting with the terminal loop of tified AREs in proto-oncogenes, transcription factors, IFNs, target precursor miRNA. and cytokines, suggesting a major contribution of ARE- Three zinc-finger RBPs were recently identified that de- http://www.jimmunol.org/ mediated decay to those genes (55). stabilize immune genes. Interaction of roquin-1 (RC3H1) with Various motifs that facilitate interaction of RBPs with the 39 UTR of Icos mRNA limits Icos expression in T cells mRNAs have been identified, including, but not limited to, (78, 79). Srivastava et al. (80) identified a novel role for roquin cytidylate uridylate–rich elements, guanylate uridylate–rich in miRNA homeostasis through direct interaction with elements, repetitive C-rich sequences, and constitutive de- Argonaute2, a central component of miRISC. They also cay elements, all of which determine mRNA stability (6). showed that roquin controls levels of miR-146a, an miRNA Classically, RBP-mediated decay begins with binding of an targeting Icos mRNA, which may explain another way in

RBP to its respective target mRNA, leading to recruitment which roquin modulates Icos. Mutation of Rc3h1 in sanroque by guest on September 27, 2021 of deadenylases to remove the poly(A) tail, followed by 39 to mice results in an autoimmune disorder caused by accumu- 59 exonucleolytic mRNA degradation, a process called ARE- lation of lymphocytes (81) and increased TNF-a levels (82). mediated decay (AMD) when it occurs via an ARE motif. Rc3h2 has a redundant role in Icos mRNA downregulation (83, RBPs also were shown to mediate 59 decapping and 59-to-39 84). Similar to roquin and tristetraprolin, regulatory RNase 1 mRNA degradation; conversely, they can also enhance mRNA (regnase-1)-deficient mice develop a systemic inflammatory stability by protecting it from other decay proteins. phenotype that is caused by increased production of IL-6 and One of the best-studied RBPs targeting AREs is triste- IL-12p40 (85). Furthermore, regnase-1 constitutively regulates traprolin. Mouse and human tristetraprolin were first de- expression of Rel, Tnfrsf4,and2 Il in naive T cells through 39 scribed in the early 1990s, as proteins encoded by the Zfp36/ UTR targeting and cleavage of mRNA (86). Upon triggering ZFP36 gene (56). Tristetraprolin and similar RBPs contain two of the TCR, the Malt1 paracaspase cleaves regnase-1 to relieve tandem repeats of zinc finger motifs that enable direct in- suppression of its targeted genes and, thus, allow timely effector teraction with AREs within the 39 UTR of target mRNAs. T cell activation and expansion (86, 87). Interestingly, although Tristetraprolin was first shown to act by recruiting the exosome regnase-1 and roquin share a specific set of target mRNAs, to ARE mRNAs to cause 39-to-59 exonucleolytic mRNA decay a recent study demonstrated differential mRNA specificity (57). However, Lykke-Andersen and Wagner (58) observed that between the two that depends on subcellular localization tristetraprolin also interacts with enzymes involved in mRNA and translational status of the respective target mRNA (88). decapping and 59-to-39 exonuclease activity, suggesting addi- The human Ag receptor (HuR) identified in 1996 (89) is tional mechanisms of tristetraprolin-mediated mRNA decay. a ubiquitously expressed member of the embryonic lethal ab- More recently, another study demonstrated that tristetraprolin normal vision–like protein family that harbors three RNA- also interacts with the CCR4–CAF1–NOT deadenylation binding domains that interact with AREs. In contrast to the complex, possibly facilitating poly(A) tail deadenylation (59). destabilizing RBPs mentioned above, HuR overexpression sta- The importance of tristetraprolin in gene regulation is evident, bilizes many ARE-containing mRNA transcripts (90, 91), 2 2 because Zfp36 / mice develop a severe systemic inflammatory including TNF (92, 93), suggesting competition between syndrome with patchy alopecia, dermatitis, erosive arthritis, RBPs for mRNA binding and a dynamic interplay of post- cachexia, conjunctivitis, myeloid hyperplasia, glomerular mesangial transcriptional regulatory elements. For example, Ogilvie et al. thickening, and antinuclear Abs within 8 wk after birth (60). (94) proposed a model of direct competition for IL2 mRNA Posttranscriptional regulation by tristetraprolin is important binding by HuR and tristetraprolin. Binding of HuR to in controlling the expression of various cytokine genes, es- the mRNA prevents tristetraprolin-mediated recruitment of the pecially inflammatory cytokines like TNF, GMCSF, IFNG, exosome to the transcript. Furthermore, dysregulation of 2966 BRIEF REVIEWS: TRANSLATING THE UNTRANSLATED REGION

HuR–tristetraprolin equilibrium was shown to drive colon carcino- Genetic variation within the HLA-C gene associates with genesis, in which loss of tristetraprolin expression and simultaneous control of HIV infection (116) and HLA-C mRNA levels, as elevated expression of HuR greatly increase tumorigenic COX-2 well as cell surface expression (117). We revealed a causal expression (95). However, in recent years, it was found that relationship between variations within the 39 UTR of HLA-C HuR can also destabilize mRNA, mainly through concerted mRNA and their effect on HIV control (118). Variation in actions with miRISC (96). Thus, the effect of HuR on mRNA the HLA-C 39 UTR determines binding by miR-148a, directly stability depends on the composite context of regulatory affecting HLA-C expression and control of HIV. The SNP elements on the 3ʹ UTR. Likewise, AU-rich element-binding (rs10889677) in the IL23R 39 UTR associates with inflam- protein 1 (AUF1) can either stabilize or destabilize immune matoryboweldisease,asitdisruptstheMREforlet-7eand gene transcripts in a cell type–dependent manner that is not let-7f, resulting in increased IL-23R expression (119). completely understood. AUF1 controls posttranscriptional SNPs can also change the secondary and tertiary structure regulation of numerous immune mediators like IL-3, IL-10, of the mRNA by changing complementarity TNF-a, and GM-CSF (97–101), as well as the miRNA pro- affecting stem-loop formation (120). Because 39 UTR stem-loops cessing machinery by targeting and repressing DICER1 (102). act as scaffolds for RBP–mRNA interactions, such changes A recent study established that AUF1 interacts with miRISC may disrupt interaction sites for RBPs and affect mRNA through Argonaute2 in either a cooperative or reciprocal stability (121). We identified a functional SNP, rs4803217, manner, depending on which target mRNA is associated; within the 39 UTR of IFNL3, which was tagged previously as this could explain, in part, the context-dependent differ- one of the stronger predictors of natural and therapy- Downloaded from ential effects of AUF1 (103). induced HCV clearance. This SNP dictates the turnover Some RBPs were shown to interact with each other or of IFNL3 mRNA by influencing the extent of AMD and compete for binding to the same recognition element, thereby miRNA-mediated decay (40). We identified the HCV- having synergistic or antagonistic effects on stability of induced miRNAs dictating IFNL3 mRNA instability (see targeted mRNA (6). For instance, roquin and regnase-1 above) and that rs4803217 allows escape of miRNA-mediated cooperate to regulate Th17 cell differentiation and expression decay to increase IFNL3 mRNA expression. However, how the http://www.jimmunol.org/ of Th17 cell–promoting factors like IL-6 and ICOS (87). The 39 UTR SNP influences IFNL3 AMD and which regulatory dynamics of such RBP interactions and their effects on gene components are involved remain unclear. expression are determined by tissue- or cell type–specific ex- Taken together, genetic variation within the 39 UTR of pression of respective RBPs, as well as by external stimuli that immune genes is a strong determinant of immune response. alter the abundance or activity of certain RBPs. Notably, triste- Sequence variations can disrupt binding sites for miRNAs traprolin levels and its extensive regulatory activity are al- and/or RBPs, altering their ability to regulate transcripts (as tered by external stimuli. To start with, the p38 MAPK summarized in Fig. 1). New sequencing technologies have

pathway is a critical regulator of tristetraprolin expression and advanced the investigations to understand these interactions by guest on September 27, 2021 activity (104, 105). MK2-mediated phosphorylation of triste- andmechanismsbehindthemanydiseasepolymorphismsin traprolin, which occurs after cytokine exposure and other noncoding regions. stress-inducing external stimuli, leads to binding of 14-3-3 proteins, exclusion of tristetraprolin from stress granules, and, Polyadenylation and 39 UTR shortening dictate mRNA function and thereby, loss of tristetraprolin-dependent suppression of turnover gene expression (106, 107). Fine-tuning of tristetraprolin Alternative polyadenylation (APA) presents a powerful mech- activity through MK2-mediated phosphorylation stabilizes anism to modulate the strength of 39 UTR–mediated regula- Tnf mRNA because it decreases tristetraprolin affinity to tion. Several polyadenylation sites can be found in a single 39 the ARE and its ability to compete with Tnf-stabilizing HuR UTR in a majority of human genes, giving rise to different for the same AREs (108). isoforms of a specific gene transcript (122, 123). APA alters The role of RBPs as immune regulators needs to be explored the length of the 39 UTR, which may affect mRNA stability further to identify additional RBPs involved in immune regu- and localization and/or translation of the isoforms (124). mRNAs lation, delineate RBP mechanisms of mRNA degradation/ with shorter 39 UTRs are generally more stable than those stabilization, and identify novel RBP–mRNA interaction motifs. with longer 39 UTRs because they encode fewer regulatory elements, allowing them to escape regulation (125). Regula- Genetic variation within the 39 UTR modulates posttranscriptional tion of APA of an mRNA transcript thereby controls mRNA regulation in disease stability and protein levels. Single-nucleotide polymorphisms (SNPs) of a gene are fre- APA was shown to be involved in B lymphocyte differen- quently associated with human diseases (40, 109–115). A tiation and the switch from membrane-bound to secreted single nucleotide change in the 39 UTR can result in dys- IgM. B cell protein levels of the essential polyadenylation regulated posttranscriptional regulation. First, alterations factor CSTF2 affect alternative processing of IgM mRNA in MRE sequences can change the affinity of the miRNA– through the differential usage of poly(A) sites (126). Low levels mRNA interaction. Functional polymorphisms located di- of CSTF2 in early-stage B cells promote cleavage at the distal rectly within destabilization motifs have been reported for IgM poly(A) site, leading to expression of membrane-bound MREs. These SNPs disrupt the interaction sites for miRNA IgM. During B cell maturation, increasing concentrations of binding, which usually leads to stabilization of the mRNA CSTF2 skew cleavage site selection toward a more proximal transcript and increased protein levels. Such miRNA target intronic poly(A) site, resulting in expression of secreted IgM. site polymorphisms are linked to immune-associated diseases A similar role for APA and involvement of CSTF2 also like cancer (114). were shown in effector T cell maturation (127). Naive T cells The Journal of Immunology 2967 Downloaded from

FIGURE 1. The posttranscriptional regulome of the 39 UTR. mRNA stability and gene expression are dictated by various posttranscriptional modulators 9

interacting with the 3 UTR. miRNAs recruit the RNA-induced silencing complex (RISC) to specific target regions, leading to RNase-mediated mRNA decay. http://www.jimmunol.org/ Similarly, instability motifs located within the 39 UTR are targeted by RBPs, resulting in rapid poly(A) tail deadenylation and mRNA degradation or stabilization. SNPs in the 39 UTR disrupt the nucleotide complementarity needed for miRNA–mRNA interactions, altering the binding capacity of miRNAs. In a similar fashion, SNPs can change the overall mRNA structure or particular instability motifs required for efficient RBP–mRNA interactions. lncRNAs are overarching modifiers of miRNA and RBP activity through their sequestration, thereby suppressing their function. Finally, shortening of the 39 UTR through usage of APA sites (pA) affects overall mRNA stability by decreasing the number of potential interaction sites/motifs for the previously mentioned posttranscriptional modulators. These regulatory elements acting in sync define the posttranscriptional regulome of the 39 UTR and ultimately dictate mRNA turnover and expression of a given gene. express two mRNA isoforms of the transcription factor Long noncoding RNAs are sinks for posttranscriptional regulators

NFATc at low levels. However, after differentiation into ef- Recently discovered long noncoding RNAs (lncRNAs) are by guest on September 27, 2021 fector T lymphocytes and a second exposure to Ags, T cells generally classified as noncoding RNAs with a size . 200 nt. rapidly start to express high amounts of a third, shorter NFATc The role of lncRNAs as overarching modifiers of gene ex- isoform. This change is caused by increased CSTF2 levels, pression became more evident in the last decade as thousands which lead to usage of a proximal APA site in the NFATc of novel lncRNA transcripts were identified by improved se- mRNA. quencing technology (134). lncRNAs interact with mRNAs, APA can also precipitate profound immune dysfunction miRNAs, and RBPs to modulate mRNA splicing, mRNA and pathology. Genetic variation within the IRF5 gene has stability, and translation, thus adding complexity to the post- been attributed to an increased risk for SLE development transcriptional regulatory mechanisms determining gene ex- (112, 128). One of three SNPs identified by Graham et al. pression and protein output. lncRNAs compete with miRNAs (129) affects IRF-5 protein levels and associates with high for binding to target mRNA, thus masking miRNA binding a risk for SLE. This SNP creates an APA signal, increasing the sites and repressing miRISC-mediated mRNA decay (135). stability of IRF5 mRNA by truncating the 39 UTR. Other Similarly, lncRNAs also interfere with miRNA-mediated gene genes such as TREX1, TLR7, and CD247 also contain variants regulation by acting as sponges to sequester miRNAs (136). in their 39 UTRs that associate with SLE risk in screened Furthermore, lncRNAs also were shown to alter mRNA sta- patient cohorts (130–132). A mutation in the single poly(A) bility by recruitment of RBPs to the 39 UTR (137). Out- site of FOXP3 mRNA was associated with development standing reviews discuss the contribution of lncRNAs to the of immune dysfunction, polyendocrinopathy, enteropathy, overall posttranscriptional regulatory machinery in more de- X-linked (110); a single A-to-G mutation in the hexameric tail (138, 139). polyadenylation signal results in impaired polyadenylation, failure to terminate transcription, and decreased FOXP3 ex- pression. In the case of proto-oncogenes, 39 UTR shortening Posttranscriptional regulome: interaction between regulators of the through APA is associated with development of cancer (133). posttranscriptional machinery dictates gene expression Thus, APA presents an efficient and powerful mechanism Rather than considering each of the above-mentioned regula- by which mRNA stability can be transiently increased within tory elements individually, we propose a more dynamic mo- a short time frame through shortening of its 39 UTR. How- del of posttranscriptional gene regulation, including synergistic ever, we still do not know how often APA is used to regulate or antagonistic interaction of several of these regulators. Jing gene expression in immune cells. New deep-sequencing tech- et al. (140) were the first to show that miRNAs and RBPs nologies will determine the extent of APA in immune genes can mediate mRNA degradation in a cooperative manner. under steady-state and activated conditions. Although this study uncovered that tristetraprolin-mediated 2968 BRIEF REVIEWS: TRANSLATING THE UNTRANSLATED REGION

TNF mRNA decay requires miR-16 processing by Dicer, as have overlooked crucial interactions between miR-29 and well as the presence of Argonaute, direct binding of miR-16 to tristetraprolin. Thus, overall, miR-29 acts as a stabilizer of tristetraprolin was not found. The investigators suggested an IFNG mRNA through its antagonism of AMD. indirect interaction of miR-16 with tristetraprolin through Another prominent example of interaction between miR- association and complex formation with Argonaute family NAs and RBPs in gene expression control is the Lin28/let-7 members (140). Later, miR-221 was found to associate with axis and its role in hematopoiesis and cancer. The RBPs tristetraprolin to facilitate TNF mRNA decay (141). Further- Lin28a and Lin28b bind to pre–let-7 to inhibit its processing more, we showed that IFNL3 mRNA stability is deter- by Drosha and Dicer (144). Lin28a/b also facilitate oligo- mined by cooperative actions of miRNAs and RBPs uridylation of pre–let-7 by TUT4, leading further to its de- targeting AREs in the IFNL3 39 UTR (40). In contrast, cay (145). Yuan et al. (146) highlighted the importance of miRNAs can also directly compete with RBPs for 39 UTR Lin28/let-7 balance in the developing immune system, when binding. For example, the seed region of miR-466l is com- they found that Lin28b was expressed in fetal, but not in plementary to the pentameric AUUUA sequence of the ARE. adult, lymphocyte progenitor cells and that Lin28b in these In the presence of miR-4661, IL10 mRNA and protein in- cells correlated inversely with expression levels of let-7 family crease (142), because miR-466l competes with tristetraprolin members. Ectopic expression of Lin28 in bone marrow he- for the AUUUA motif and prevents tristetraprolin-mediated matopoietic stem/progenitor cells represses let-7 miRNAs and degradation. miR-29 also was identified as a stabilizer of tu- allows multilineage reconstitution. mor suppressor A20 mRNA through interaction with other Many examples given in previous sections of this review Downloaded from posttranscriptional elements, where it acts as a RNA decoy for combine diverse simultaneous posttranscriptional regulatory the RBP HuR, thus preventing A20 mRNA decay (143). elements, which can either compete or cooperate. Given this Additionally, we uncovered a similar mechanism by which ample evidence that the interplay of multiple factors can miR-29 stabilizes gene expression of IFNG through com- result in a vastly different phenotype from the actions of in- petitive binding to RBP-recognition elements. To study dividual components, integrating these regulatory pathways thesimultaneouseffectsofmiR-29andAREsonIFNG while studying these regulations will provide a complete picture http://www.jimmunol.org/ mRNA stability, we retained RNA structural integrity by of the 39 UTR “regulome” of the gene. using constructs containing the complete 39 UTR sequence. We observed that miR-29 stabilizes IFNG expression in the Conclusions presence of the tristetraprolin complex (R. Savan and Posttranscriptional regulation through the 39 UTR is a potent H.A. Young, unpublished observations). The AREs targeted mechanism to control the development and homeostasis of by tristetraprolin for mRNA decay are in close proximity to the immune system and to quickly adjust the expression the miR-29 binding site in the secondary mRNA structure. of immune genes upon stimulation by cell-intrinsic or cell-

We propose that this prevents the recruitment of GW182 so extrinsic cues. In this review, we discussed different mech- by guest on September 27, 2021 that miRISC cannot degrade the transcript. Although miR-29 anisms by which posttranscriptional regulation through the was shown to enhance degradation of IFNG (32), these studies 39 UTR of mRNA transcripts occurs. miRISC and AMD are used partial UTR sequences lacking the AREs and, thus, may the most powerful regulatory elements dictating mRNA

FIGURE 2. Posttranscriptional reg- ulation of the 39 UTR and its implica- tions for immune disorders. Insertion (INS) or deletion (DEL) mutations in the 39 UTR of the gene create or destroy binding sites for regulators, such as RBPs and miRNAs. This leads to in- creased or decreased mRNA stability and protein levels. Likewise, SNPs in the 39 UTR can create new APA signals or destroy poly(A) signals, altering the length of the 39 UTR affecting the sta- bility of mRNA. Elimination of interac- tion motifs/sequences for RBPs and miRNAs leads to increased mRNA stability and protein levels. These SNPs affect posttranscriptional control of genes involved in innate and adap- tive immunity. Hence, carriers of the risk or protective alleles can be more likely to develop or resist immune disorders like immunodeficiency, auto- immunity, or cancer. The Journal of Immunology 2969 stability and/or degradation. 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