Roquin Suppresses the PI3K-mTOR Signaling Pathway to Inhibit T Helper Cell Differentiation and Conversion of Treg to Tfr Cells.

Roquin proteins preclude spontaneous T cell activation and aberrant differentiation of T follicular helper (Tfh) or T helper 17 (Th17) cells. Here we showed that deletion of Roquin-encoding alleles specifically in regulatory T (Treg) cells also caused the activation of conventional T cells. Roquin-deficient Treg cells downregulated CD25, acquired a follicular Treg (Tfr) cell phenotype, and suppressed germinal center reactions but could not protect from colitis. Roquin inhibited the PI3K-mTOR signaling pathway by upregulation of Pten through interfering with miR-17∼92 binding to an overlapping cis-element in the Pten 3' UTR, and downregulated the Foxo1-specific E3 ubiquitin ligase Itch. Loss of Roquin enhanced Akt-mTOR signaling and protein synthesis, whereas inhibition of PI3K or mTOR in Roquin-deficient T cells corrected enhanced Tfh and Th17 or reduced iTreg cell differentiation. Thereby, Roquin-mediated control of PI3K-mTOR signaling prevents autoimmunity by restraining activation and differentiation of conventional T cells and specialization of Treg cells.

A temporal difference in the stabilization of two mRNAs with a 3' iron-responsive element during iron deficiency.

The interactions of iron regulatory proteins (IRPs) with mRNAs containing an iron-responsive element (IRE) maintain cellular iron homeostasis and coordinate it with metabolism and possibly cellular behavior. The mRNA encoding transferrin receptor-1 (TFRC, TfR1), which is a major means of iron importation, has five IREs within its 3' UTR, and IRP interactions help maintain cytosolic iron through the protection of the TfR1 mRNA from degradation. An IRE within the 3' UTR of an mRNA splice variant encoding human cell division cycle 14A (CDC14A) has the potential to coordinate the cellular iron status with cellular behavior through a similar IRP-mediated mechanism. However, the stability of the CDC14A splice variant was reported earlier to be unaffected by the cellular iron status, which suggested that the IRE is not functional. We labeled newly synthesized mRNA in HEK293 cells with 5-ethynyl uridine and found that the stability of the CDC14A variant is responsive to iron deprivation, but there are two major differences from the regulation of TfR1 mRNA stability. First, the decay of the CDC14A mRNA does not utilize the Roquin-mediated reaction that acts on the TfR1 mRNA, indicating that there is flexibility in the degradative machinery antagonized by the IRE-IRP interactions. Second, the stabilization of the CDC14A mRNA is delayed relative to the TfR1 mRNA and does not occur until IRP binding activity has been induced. The result is consistent with a hierarchy of IRP interactions in which the maintenance of cellular iron through the stabilization of the TfR1 mRNA is initially prioritized.

Bioinformatic Analysis of Roquin Family Reveals Their Potential Role in Immune System.

The Roquin family is a recognized RNA-binding protein family that plays vital roles in regulating the expression of pro-inflammatory target gene mRNA during the immune process in mammals. However, the evolutionary status of the Roquin family across metazoans remains elusive, and limited studies are found in fish species. In this study, we discovered that the RC3H genes underwent a single round of gene duplication from a primitive ancestor during evolution from invertebrates to vertebrates. Furthermore, there were instances of species-specific gene loss events or teleost lineage-specific gene duplications throughout evolution. Domain/motif organization and selective pressure analysis revealed that Roquins exhibit high homology both within members of the family within the same species and across species. The three rc3h genes in zebrafish displayed similar expression patterns in early embryos and adult tissues, with rc3h1b showing the most prominent expression among them. Additionally, the promoter regions of the zebrafish rc3h genes contained numerous transcription factor binding sites similar to those of mammalian homologs. Moreover, the interaction protein network of Roquin and the potential binding motif in the 3'-UTR of putative target genes analysis both indicated that Roquins have the potential to degrade target mRNA through mechanisms similar to those of mammalian homologs. These findings shed light on the evolutionary history of Roquin among metazoans and hypothesized their role in the immune systems of zebrafish.

Discovery of Small-Molecule Modulators of Protein-RNA Interactions by Fluorescence Intensity-Based Binding Assay.

Protein-RNA interactions mediate various cellular processes, the dysregulation of which has been associated with a list of diseases. Thus, novel experimental tools for monitoring protein-RNA interactions are highly desirable to identify new chemical modulators of these therapeutic targets. In this study, we constructed simple fluorescence intensity-based protein-RNA binding assays by testing multiple environment-sensitive organic fluorophores. We selected the oncogenic interaction between Lin28 and the let-7 microRNA and the important immunomodulatory Roquin-Tnf CDE interaction as representative targets. We adapted this assay to high-throughput screening for the identification of pyrazolyl thiazolidinedione-type molecules as potent small-molecule inhibitors of protein-microRNA interactions. We clearly showed the structure-activity relationships of this new class of Lin28-let-7 interaction inhibitors, and confirmed that cellular mature let-7 microRNAs and their target genes could be modulated upon treatment with the pyrazolyl thiazolidinedione-type inhibitor. We expect that our simple and adaptable screening approach can be applied for the development of various assay systems aimed at the identification of bioactive small molecules targeting protein-RNA interactions.

RNA-binding protein Roquin negatively regulates STING-dependent innate immune response in Drosophila.

Drosophila melanogaster utilizes innate immune response to defend against exogenous pathogens. The molecular regulation mechanism of the process is evolutionarily conserved. Research of the regulatory mechanisms of Drosophila innate immunity is greatly significant for understanding the modulation of the human innate immunity and the pathogenesis of related diseases. To explore novel regulators in the STING-dependent innate immune response in Drosophila, we utilized the double-stranded RNA-mediated gene expression silencing technique and the dual-luciferase reporter system in knockdown experiments on 9 genes encoding the ubiquitin ligase such as echinus (CG2904), usp16 (CG4165), smurf (CG4943), pellino (CG5212), usp47 (CG5486), diap2 (CG8293), dtraf2 (CG10961), roquin (CG16807) and usp10 (CG32479) in the S2 cells in vitro. The results suggested a negative correlation between CG16807 (roquin) and the STING signaling pathway. Further studies showed that over-expression of roquin in S2 cells significantly inhibited STING innate immune signaling. Meanwhile, Listeria infection experiments showed that knocking down of roquin markedly elevated the expression levels of anti-microbial peptides and inhibited the proliferation of Listeria, thus increasing the survival rates post pathogenic infection. Taken together, our results suggested that the RNA-binding protein Roquin negatively regulates the STING-dependent innate immune response in Drosophila. In view of the high correlation between Drosophila genes and human genes, this study provides a theoretical basis for further development of treatments for STING-related innate immune diseases in humans.

Exaptive origins of regulated mRNA decay in eukaryotes.

Eukaryotic gene expression is extensively controlled at the level of mRNA stability and the mechanisms underlying this regulation are markedly different from their archaeal and bacterial counterparts. We propose that two such mechanisms, nonsense-mediated decay (NMD) and motif-specific transcript destabilization by CCCH-type zinc finger RNA-binding proteins, originated as a part of cellular defense against RNA pathogens. These branches of the mRNA turnover pathway might have been used by primeval eukaryotes alongside RNA interference to distinguish their own messages from those of RNA viruses and retrotransposable elements. We further hypothesize that the subsequent advent of "professional" innate and adaptive immunity systems allowed NMD and the motif-triggered mechanisms to be efficiently repurposed for regulation of endogenous cellular transcripts. This scenario explains the rapid emergence of archetypical mRNA destabilization pathways in eukaryotes and argues that other aspects of post-transcriptional gene regulation in this lineage might have been derived through a similar exaptation route.

ROQUIN signalling pathways in innate and adaptive immunity.

ROQUIN is an RNA-binding protein that plays important roles in both the innate and adaptive immune systems. ROQUIN binds to several key immune-relevant messenger RNA (mRNA) targets through its ROQ domain modulating their stability and influencing macrophage function and the peripheral homeostasis of T cells and B cells. More recently, the E3 ubiquitin ligase activity of the ROQUIN RING domain has been shown to be crucial for T-cell-dependent B-cell responses against infection. Defective ROQUIN activity can culminate in a range of diseases, such as systemic autoimmunity, immunodeficiency, and inflammatory bowel disorder. Here, we provide a current overview of the immunomodulatory role of ROQUIN defined by its ribonucleoprotein-like structure, its repertoire of mRNA targets shared by related RNA-binding enzymes, and its involvement in a range of intracellular signalling pathways central to shaping immune responses.

Involvement of Ly6C, 4-1BB, and KLRG1 in the activation of lamina propria lymphocytes in the small intestine of sanroque mice.

Roquin is an E3 ligase that regulates mRNA stability. Mice with a mutation in the Rc3h1 gene and Roquin protein, referred to as Roquinsan/san or sanroque mice, develop broad-spectrum chronic inflammatory conditions and autoimmune pathologies. Our laboratory recently reported that sanroque mice also develop extensive inflammation that is localized in the small intestine but is rare in the colon. Here, we demonstrate that small intestinal intraepithelial lymphocytes (IELs) are present in the epithelium of sanroque mice but that cell recoverability is low using standard extraction techniques even though lamina propria lymphocytes (LPLs) can be recovered in normal numbers. In studies aimed at characterizing T cell costimulatory markers and activation molecules on LPLs in sanroque mice, we identified Ly6C and 4-1BB (CD137) as being expressed at elevated levels on sanroque small intestinal LPLs, and we show that both of those subsets, in conjunction with cells expressing the KLRG1 T cell activation molecule, are sources of IL-17A, IFN-γ, and TNFα. TNFα was primarily produced by 4-1BB+, KLRG1-cells, but was also made by some 4-1BB-, KLRG1-cells, and 4-1BB-, KLRG1+ cells. These findings collectively suggest that the small intestinal inflammatory response in sanroque mice is driven, at least in part, by LPL activation through Ly6C and 4-1BB signaling, and they provide further evidence in support of using the sanroque mouse as an animal model of chronic small intestinal inflammation.

Over-expression of Roquin aggravates T cell mediated hepatitis in transgenic mice using T cell specific promoter.

Chronic hepatitis is a major cause of liver cancer, so earlier treatment of hepatitis might be reducing liver cancer incidence. Hepatitis can be induced in mice by treatment with Concanavalin A (Con A); the resulting liver injury causes significant CD4(+) T cell activation and infiltration. In these T cells, Roquin, a ring-type E3 ubiquitin ligase, is activated. To investigate the role of Roquin, we examined Con A-induced liver injury and T cell infiltration in transgenic (Tg) mice overexpressing Roquin specifically in T cells. In Roquin Tg mice, Con A treatment caused greater increases in both the levels of liver injury enzymes and liver tissue apoptosis, as revealed by TUNEL and H&E staining, than wild type (WT) mice. Further, Roquin Tg mice respond to Con A treatment with greater increases in the T cell population, particularly Th17 cells, though Treg cell counts are lower. Roquin overexpression also enhances increases in pro-inflammatory cytokines, including IFN-γ, TNF-α and IL-6, upon liver injury. Furthermore, Roquin regulates the immune response and apoptosis in Con A induced hepatitis via STATs, Bax and Bcl2. These findings suggest that over-expression of Roquin exacerbates T-cell mediated hepatitis.

Roquin-2 promotes oxidative stress-induced cell death by ubiquitination-dependent degradation of TAK1.

Reactive oxygen species (ROS) are highly reactive and their accumulation causes oxidative damage to cells. Cells maintain survival upon mild oxidative stress with anti-oxidative systems, such as the kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) system. On the other hand, upon severe oxidative stress, cells undergo regulated cell death, including apoptosis, for eliminating damaged cells. To execute efficient cell death, cells need to turn off the anti-oxidant systems, while triggering cell death. However, it remains unknown how cells orchestrate these two conflicting systems under excessive oxidative stress. Herein, we show that when cells are exposed to excessive oxidative damage, an E3 ubiquitin ligase Roquin-2 (also known as RC3H2) plays a key role in switching cell fate from survival to death by terminating activation of transforming growth factor-ß-activated kinase 1 (TAK1), a positive regulator for Nrf2 activation. Roquin-2 interacted with TAK1 via four cysteine residues in TAK1 (C96, C302, C486, and C500) that are susceptible to oxidative stress and participate in oligomer formation via disulfide bonds, promoting K48-linked polyubiquitination and degradation of TAK1. Nrf2 was inactivated upon lethal oxidative stress in wild-type mouse embryonic fibroblast (MEF) cells, whereas it sustained activation and conferred resistance to Roquin-2 deficient cells, which was reversed by pharmacological or genetic inhibition of TAK1. These data demonstrate that in response to excessive ROS exposure, Roquin-2 promotes ubiquitination and degradation of TAK1 to suppress Nrf2 activation, and thereby contributes to an efficient cell death, providing insight into the pathogenesis of oxidative stress-related diseases, including cancer.

Analysis of ROQUIN, Tristetraprolin (TTP), and BDNF/miR-16/TTP regulatory axis in late onset Alzheimer's disease.

Alzheimer's disease (AD) is a heterogeneous degenerative disorder of the brain that is on the rise worldwide. One of the critical processes that might be disturbed in AD is gene expression regulation. Tristetraprolin (TTP) and RC3H1 gene (ROQUIN) are two RNA-binding proteins (RBPs) that target AU-rich elements (AREs) and constitutive decay elements (CDEs), respectively. TTP and ROQUIN, members of the CCCH zinc-finger protein family, have been demonstrated to fine-tune numerous inflammatory factors. In addition, miR-16 has distinct characteristics and may influence the target mRNA through the ARE site. Interestingly, BDNF mRNA has ARE sites in the 3' untranslated region (UTR) and can be targeted by regulatory factors, such as TTP and miR-16 on MRE sequences, forming BDNF/miR-16/TTP regulatory axis. A number of two microarray datasets were downloaded, including information on mRNAs (GSE106241) and miRNAs (GSE157239) from individuals with AD and corresponding controls. R software was used to identify BDNF, TTP, ROQUIN, and miR-16 expression levels in temporal cortex (TC) tissue datasets. Q-PCR was also used to evaluate the expression of these regulatory factors and the expression of BDNF in the blood of 50 patients with AD and 50 controls. Bioinformatic evaluation showed that TTP and miR-16 overexpression might act as post-transcriptional regulatory factors to control BDNF expression in AD in TC samples. Instead, this expression pattern was not found in peripheral blood samples from patients with AD compared to normal controls. ROQUIN expression was increased in the peripheral blood of patients with AD. Hsa-miR-16-5p levels did not show significant differences in peripheral blood samples. Finally, it was shown that TTP and BDNF, based on evaluating the receiver operating characteristic (ROC), effectively identify patients with AD from healthy controls. This study could provide a new perspective on the molecular regulatory processes associated with AD pathogenic mechanisms linked to the BDNF growth factor, although further research is needed on the possible roles of these factors in AD.

Cooperation of RNA-Binding Proteins - a Focus on Roquin Function in T Cells.

Post-transcriptional gene regulation by RNA-binding proteins (RBPs) is important in the prevention of inflammatory and autoimmune diseases. With respect to T cell activation and differentiation, the RBPs Roquin-1/2 and Regnase-1 play pivotal roles by inducing degradation and/or translational silencing of target mRNAs. These targets encode important proinflammatory mediators and thus Roquin and Regnase-1 functions dampen cellular programs that can lead to inflammation and autoimmune disease. Recent findings demonstrate direct physical interaction of both RBPs. Here, we propose that cooperativity of trans-acting factors may be more generally used to reinforce the regulatory impact on selected targets and promote specific cell fate decisions. We develop this concept for Roquin and Regnase-1 function in resting and activated T cells and discuss the involvement in autoimmunity as well as how the therapeutic potential can be used in anti-tumor therapies.

Roquin2 suppresses breast cancer progression by inhibiting tumor angiogenesis via selectively destabilizing proangiogenic factors mRNA.

Tumor angiogenesis is an essential step in tumor growth and metastasis. The initiation of tumor angiogenesis is dictated by a shift in the balance between proangiogenic and antiangiogenic gene expression programs. Roquin2 is a zinc-finger RNA-binding protein with important roles in mediating the expression of inflammatory genes, such as TNF, IL6 and PTGS2, which are also important angiogenic factors. In this study, we demonstrate that Roquin2 functions as a potent tumor angiogenesis regulator that inhibits breast tumor-induced angiogenesis by selectively destabilizing mRNA of proangiogenic gene transcripts, including endoglin (ENG), endothelin-1 (EDN1), vascular endothelial growth factor B (VEGFB) and platelet derived growth factor C (PDGFC). Roquin2 recognizes and binds the stem-loop structure in the 3'untranslated region (3'UTR) of these mRNAs via its ROQ domain to destabilize mRNA. Moreover, we found that Roquin2 expression was reduced in breast cancer cells and tissues, and associated with poor prognosis in breast cancer patients. Overexpression of Roquin2 inhibited breast tumor-induced angiogenesis in vitro and in vivo, whereas silencing Roquin2 enhanced tumor angiogenesis. In vivo induction of Roquin2 by adenovirus significantly suppressed breast tumor growth, metastasis and angiogenesis. Taken together, our results identify that Roquin2 is a novel breast cancer suppressor that inhibits tumor angiogenesis by selectively downregulating the expression of proangiogenic genes.

Genome-wide fitness gene identification reveals Roquin as a potent suppressor of CD8 T cell expansion and anti-tumor immunity.

Robust expansion of adoptively transferred T cells is a prerequisite for effective cancer immunotherapy, but how many genes in the genome modulate T cell expansion remains unknown. Here, we perform in vivo and in vitro CRISPR screens to systematically identify genes influencing CD8 T cell expansion. In the mouse genome, ∼2,600 and ∼1,500 genes are required for optimal CD8 T cell expansion in vivo and in vitro, respectively. In vivo-specific CD8 T cell essential genes are enriched in metabolic pathways, including mitochondrial metabolism. The strongest repressor of CD8 T cell expansion is Roquin, the ablation of which drastically boosts T cell proliferation by enhancing cell-cycle progression and upregulation of IRF4. Roquin deficiency or IRF4 overexpression potently enhances anti-tumor immunity. These data provide a functional catalog of CD8 T cell fitness genes and suggest that targeting the Roquin-IRF4 axis is an effective strategy to enhance efficacy of adoptive transfer therapy for cancer.

Roquin1 inhibits the proliferation of breast cancer cells by inducing G1/S cell cycle arrest via selectively destabilizing the mRNAs of cell cycle-promoting genes.

BACKGROUND: Dysregulation of cell cycle progression is a common feature of human cancer cells; however, its mechanism remains unclear. This study aims to clarify the role and the underlying mechanisms of Roquin1 in cell cycle arrest in breast cancer. METHODS: Public cancer databases were analyzed to identify the expression pattern of Roquin1 in human breast cancers and its association with patient survival. Quantitative real-time PCR and Western blots were performed to detect the expression of Roquin1 in breast cancer samples and cell lines. Cell counting, MTT assays, flow cytometry, and in vivo analyses were conducted to investigate the effects of Roquin1 on cell proliferation, cell cycle progression and tumor progression. RNA sequencing was applied to identify the differentially expressed genes regulated by Roquin1. RNA immunoprecipitation assay, luciferase reporter assay, mRNA half-life detection, RNA affinity binding assay, and RIP-ChIP were used to explore the molecular mechanisms of Roquin1. RESULTS: We showed that Roquin1 expression in breast cancer tissues and cell lines was inhibited, and the reduction in Roquin1 expression was associated with poor overall survival and relapse-free survival of patients with breast cancer. Roquin1 overexpression inhibited cell proliferation and induced G1/S cell cycle arrest without causing significant apoptosis. In contrast, knockdown of Roquin1 promoted cell growth and cycle progression. Moreover, in vivo induction of Roquin1 by adenovirus significantly suppressed breast tumor growth and metastasis. Mechanistically, Roquin1 selectively destabilizes cell cycle-promoting genes, including Cyclin D1, Cyclin E1, cyclin dependent kinase 6 (CDK6) and minichromosome maintenance 2 (MCM2), by targeting the stem-loop structure in the 3' untranslated region (3'UTR) of mRNAs via its ROQ domain, leading to the downregulation of cell cycle-promoting mRNAs. CONCLUSIONS: Our findings demonstrated that Roquin1 is a novel breast tumor suppressor and could induce G1/S cell cycle arrest by selectively downregulating the expression of cell cycle-promoting genes, which might be a potential molecular target for breast cancer treatment.

KLHL6 is a tumor suppressor gene in diffuse large B-cell lymphoma.

The ubiquitin proteasome system (UPS) plays a critical function in cellular homeostasis. The misregulation of UPS is often found in human diseases, including cancer. Kelch-like protein 6 (KLHL6) is an E3 ligase gene mutated in diffused large B-cell lymphoma (DLBCL). This review discusses the function of KLHL6 as a cullin3-RING ligase and how cancer-associated mutations disrupt the interaction with the cullin3, resulting in the loss of KLHL6 function. Furthermore, the mRNA decay factor Roquin2 is discussed as the first bona fide substrate of KLHL6 in the context of B-cell receptor activation and B-cell lymphoma. Importantly, the tumor-suppressing mechanism of KLHL6 via the degradation of Roquin2 and the mRNA decay in the context of the NF-κB pathway is summarized.

RNA binding proteins in the control of autoimmune diseases.

Autoimmune disease is induced by the breakdown of immune tolerance to self-antigens. This is brought about by an imbalance between the activation and the repression of immune responses. Dysregulation of the immune response is driven by the excess of proinflammatory cytokines such as IL-6 and TNF, which play a central role in the pathogenesis of a set of autoimmune diseases. The expression of proinflammatory mediator genes is tightly controlled by post-transcriptional regulation, which is mediated by a set of immune-related RNA binding proteins, such as tristetraprolin, Roquin, and Regnase-1. These proteins coordinately control the stability of proinflammatory mRNAs to regulate aberrant immune reactions. In this review, we discuss the roles of RNA binding proteins which are associated with the immune regulation and autoimmune pathogenesis.

Posttranscriptional Gene Regulation of T Follicular Helper Cells by RNA-Binding Proteins and microRNAs.

T follicular helper (Tfh) cells are critically involved in the establishment of potent antibody responses against infectious pathogens, such as viruses and bacteria, but their dysregulation may also result in aberrant antibody responses that frequently coincide with autoimmune diseases or allergies. The fate and identity of Tfh cells is tightly controlled by gene regulation on the transcriptional and posttranscriptional level. Here, we provide deeper insights into the posttranscriptional mechanisms that regulate Tfh cell differentiation, function, and plasticity through the actions of RNA-binding proteins (RBPs) and small endogenously expressed regulatory RNAs called microRNAs (miRNAs). The Roquin family of RBPs has been shown to dampen spontaneous activation and differentiation of naïve CD4+ T cells into Tfh cells, since CD4+ T cells with Roquin mutations accumulate as Tfh cells and provide inappropriate B cell help in the production of autoantibodies. Moreover, Regnase-1, an endoribonuclease that regulates a set of targets, which strongly overlaps with that of Roquin, is crucial for the prevention of autoantibody production. Interestingly, both Roquin and Regnase-1 proteins are cleaved and inactivated after TCR stimulation by the paracaspase MALT1. miRNAs are expressed in naïve CD4+ T cells and help preventing spontaneous differentiation into effector cells. While most miRNAs are downregulated upon T cell activation, several miRNAs have been shown to regulate the fate of these cells by either promoting (e.g., miR-17-92 and miR-155) or inhibiting (e.g., miR-146a) Tfh cell differentiation. Together, these different aspects highlight a complex and dynamic regulatory network of posttranscriptional gene regulation in Tfh cells that may also be active in other T helper cell populations, including Th1, Th2, Th17, and Treg.

Structure of human Roquin-2 and its complex with constitutive-decay element RNA.

Roquin mediates mRNA degradation by recognizing the constitutive-decay element (CDE) in the 3' untranslated region of the target gene followed by recruitment of the deadenylation machinery. Deficiency or dysfunction of Roquin has been associated with autoimmunity and inflammation. To establish the structural basis for the recognition of CDE RNA by Roquin, the crystal structure of the ROQ domain of human Roquin-2 was determined in ligand-free and CDE-derived RNA-bound forms. The ROQ domain of Roquin-2 folded into a winged-helix structure in which the wing region showed structural flexibility and acted as a lid for RNA binding. The CDE RNA, forming a stem-loop structure, bound to the positively charged surface of the ROQ domain and was mainly recognized via direct interactions with the phosphate backbone in the 5' half of the stem-loop and its triloop and via indirect water-mediated interactions. Structural comparison with Roquin-1 revealed conserved features of the RNA-binding mode. Therefore, it is suggested that the Roquin proteins function redundantly in mRNA degradation.

Attenuation of AMPK signaling by ROQUIN promotes T follicular helper cell formation.

T follicular helper cells (Tfh) are critical for the longevity and quality of antibody-mediated protection against infection. Yet few signaling pathways have been identified to be unique solely to Tfh development. ROQUIN is a post-transcriptional repressor of T cells, acting through its ROQ domain to destabilize mRNA targets important for Th1, Th17, and Tfh biology. Here, we report that ROQUIN has a paradoxical function on Tfh differentiation mediated by its RING domain: mice with a T cell-specific deletion of the ROQUIN RING domain have unchanged Th1, Th2, Th17, and Tregs during a T-dependent response but show a profoundly defective antigen-specific Tfh compartment. ROQUIN RING signaling directly antagonized the catalytic α1 subunit of adenosine monophosphate-activated protein kinase (AMPK), a central stress-responsive regulator of cellular metabolism and mTOR signaling, which is known to facilitate T-dependent humoral immunity. We therefore unexpectedly uncover a ROQUIN-AMPK metabolic signaling nexus essential for selectively promoting Tfh responses.