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Activation induces extensive changes in the gene expression program of naive CD4+ T cells, promoting their differentiation into helper T cells that coordinate immune responses. MicroRNAs (miRNAs) play a critical role in this process, and miRNA expression a

Caspase-11 is a key regulator of proinflammatory cytokine IL-1ß maturation and pathological apoptosis. Caspase-11 is not expressed in most tissues under normal condition, but highly inducible upon pathological stimulation such as in the presence of lipopolysaccharide (LPS). Here, we describe the identification and characterization of wedelolactone, a natural compound that inhibits LPS-induced caspase-11 expression in cultured cells by inhibiting NF-?B-mediated transcription. We demonstrate that wedelolactone is an inhibitor of IKK, a kinase critical for activation of NF-?B by mediating phosphorylation and degradation of I?Ba.Cell Death and Differentiation (2004) 11, 123-130. doi:10.1038/sj.cdd.4401325 Published online 3 October 2003 [ABSTRACT FROM AUTHOR]

Aloperine (ALO), a quinolizidine-type alkaloid isolated from a natural Chinese herb, has shown promising antitumor effects. Nevertheless, its common mechanism of action and specific target remain elusive. Here, it is demonstrated that ALO inhibits the proliferation and migration of non-small cell lung cancer cell lines in vitro and the tumor development in several mouse tumor models in vivo. Mechanistically, ALO inhibits the fusion of autophagosomes with lysosomes and the autophagic flux, leading to the accumulation of sequestosome-1 (SQSTM1) and production of reactive oxygen species (ROS), thereby inducing tumor cell apoptosis and preventing tumor growth. Knockdown of SQSTM1 in cells inhibits ROS production and reverses ALO-induced cell apoptosis. Furthermore, VPS4A is identified as a direct target of ALO, and the amino acids F153 and D263 of VPS4A are confirmed as the binding sites for ALO. Knockout of VPS4A in H1299 cells demonstrates a similar biological effect as ALO treatment. Additionally, ALO enhances the efficacy of the anti-PD-L1/TGF-β bispecific antibody in inhibiting LLC-derived subcutaneous tumor models. Thus, ALO is first identified as a novel late-stage autophagy inhibitor that triggers tumor cell death by targeting VPS4A., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Post-transcriptional regulation of immune-related transcripts by RNA-binding proteins (RBPs) impacts immune cell responses, including mast cell functionality. Despite their importance in immune regulation, the functional role of most RBPs remains to be understood. By manipulating the expression of specific RBPs in murine mast cells, coupled with mass spectrometry and transcriptomic analyses, we found that the Regnase family of proteins acts as a potent regulator of mast cell physiology. Specifically, Regnase-1 is required to maintain basic cell proliferation and survival, whereas both Regnase-1 and -3 cooperatively regulate the expression of inflammatory transcripts upon activation, with Tnf being a primary target in both human and mouse cells. Furthermore, Regnase-3 directly interacts with Regnase-1 in mast cells and is necessary to restrain Regnase-1 expression through the destabilization of its transcript. Overall, our study identifies protein interactors of endogenously expressed Regnase factors, characterizes the regulatory interplay between Regnase family members in mast cells, and establishes their role in the control of mast cell homeostasis and inflammatory responses., (© 2024 Bataclan et al.)

The regulation of thymocyte development by RNA-binding proteins (RBPs) is largely unexplored. We identify 642 RBPs in the thymus and focus on Arpp21, which shows selective and dynamic expression in early thymocytes. Arpp21 is downregulated in response to T cell receptor (TCR) and Ca 2+ signals. Downregulation requires Stim1/Stim2 and CaMK4 expression and involves Arpp21 protein phosphorylation, polyubiquitination and proteasomal degradation. Arpp21 directly binds RNA through its R3H domain, with a preference for uridine-rich motifs, promoting the expression of target mRNAs. Analysis of the Arpp21-bound transcriptome reveals strong interactions with the Rag1 3'-UTR. Arpp21-deficient thymocytes show reduced Rag1 expression, delayed TCR rearrangement and a less diverse TCR repertoire. This phenotype is recapitulated in Rag1 3'-UTR mutant mice harboring a deletion of the Arpp21 response region. These findings show how thymocyte-specific Arpp21 promotes Rag1 expression to enable TCR repertoire diversity until signals from the TCR terminate Arpp21 and Rag1 activities., (© 2024. The Author(s).)

T follicular helper (T FH ) cells are essential for effective antibody responses, but deciphering the intrinsic wiring of mouse T FH cells has long been hampered by the lack of a reliable protocol for their generation in vitro. We report that transforming growth factor-β (TGF-β) induces robust expression of T FH hallmark molecules CXCR5 and Bcl6 in activated mouse CD4 + T cells in vitro. TGF-β-induced mouse CXCR5 + T FH cells are phenotypically, transcriptionally, and functionally similar to in vivo-generated T FH cells and provide critical help to B cells. The study further reveals that TGF-β-induced CXCR5 expression is independent of Bcl6 but requires the transcription factor c-Maf. Classical TGF-β-containing T helper 17 (T H 17)-inducing conditions also yield separate CXCR5 + and IL-17A-producing cells, highlighting shared and distinct cell fate trajectories of T FH and T H 17 cells. We demonstrate that excess IL-2 in high-density T cell cultures interferes with the TGF-β-induced T FH cell program, that T FH and T H 17 cells share a common developmental stage, and that c-Maf acts as a switch factor for T FH versus T H 17 cell fates in TGF-β-rich environments in vitro and in vivo.

Constitutive activation of the MALT1 paracaspase in conventional T cells of Malt1 TBM/TBM (TRAF6 Binding Mutant = TBM) mice causes fatal inflammation and autoimmunity, but the involved targets and underlying molecular mechanisms are unknown. We genetically rendered a single MALT1 substrate, the RNA-binding protein (RBP) Roquin-1, insensitive to MALT1 cleavage. These Rc3h1 Mins/Mins mice showed normal immune homeostasis. Combining Rc3h1 Mins/Mins alleles with those encoding for constitutively active MALT1 (TBM) prevented spontaneous T cell activation and restored viability of Malt1 TBM/TBM mice. Mechanistically, we show how antigen/MHC recognition is translated by MALT1 into Roquin cleavage and derepression of Roquin targets. Increasing T cell receptor (TCR) signals inactivated Roquin more effectively, and only high TCR strength enabled derepression of high-affinity targets to promote Th17 differentiation. Induction of experimental autoimmune encephalomyelitis (EAE) revealed increased cleavage of Roquin-1 in disease-associated Th17 compared to Th1 cells in the CNS. T cells from Rc3h1 Mins/Mins mice did not efficiently induce the high-affinity Roquin-1 target IκB NS in response to TCR stimulation, showed reduced Th17 differentiation, and Rc3h1 Mins/Mins mice were protected from EAE. These data demonstrate how TCR signaling and MALT1 activation utilize graded cleavage of Roquin to differentially regulate target mRNAs that control T cell activation and differentiation as well as the development of autoimmunity., Competing Interests: Competing interests statement:The authors declare no competing interest.

ERI1 is a 3'-to-5' exoribonuclease involved in RNA metabolic pathways including 5.8S rRNA processing and turnover of histone mRNAs. Its biological and medical significance remain unclear. Here, we uncover a phenotypic dichotomy associated with bi-allelic ERI1 variants by reporting eight affected individuals from seven unrelated families. A severe spondyloepimetaphyseal dysplasia (SEMD) was identified in five affected individuals with missense variants but not in those with bi-allelic null variants, who showed mild intellectual disability and digital anomalies. The ERI1 missense variants cause a loss of the exoribonuclease activity, leading to defective trimming of the 5.8S rRNA 3' end and a decreased degradation of replication-dependent histone mRNAs. Affected-individual-derived induced pluripotent stem cells (iPSCs) showed impaired in vitro chondrogenesis with downregulation of genes regulating skeletal patterning. Our study establishes an entity previously unreported in OMIM and provides a model showing a more severe effect of missense alleles than null alleles within recessive genotypes, suggesting a key role of ERI1-mediated RNA metabolism in human skeletal patterning and chondrogenesis., Competing Interests: Declaration of interests The Department of Molecular and Human Genetics at Baylor College of Medicine receives revenue from clinical genetic testing completed at Baylor Genetics Laboratories., (Copyright © 2023 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Mast cells are central players in allergy and asthma, and their dysregulated responses lead to reduced quality of life and life-threatening conditions such as anaphylaxis. The RNA modification N 6 -methyladenosine (m 6 A) has a prominent impact on immune cell functions, but its role in mast cells remains unexplored. Here, by optimizing tools to genetically manipulate primary mast cells, we reveal that the m 6 A mRNA methyltransferase complex modulates mast cell proliferation and survival. Depletion of the catalytic component Mettl3 exacerbates effector functions in response to IgE and antigen complexes, both in vitro and in vivo. Mechanistically, deletion of Mettl3 or Mettl14, another component of the methyltransferase complex, lead to the enhanced expression of inflammatory cytokines. By focusing on one of the most affected mRNAs, namely the one encoding the cytokine IL-13, we find that it is methylated in activated mast cells, and that Mettl3 affects its transcript stability in an enzymatic activity-dependent manner, requiring consensus m 6 A sites in the Il13 3'-untranslated region. Overall, we reveal that the m 6 A machinery is essential in mast cells to sustain growth and to restrain inflammatory responses., (© 2023. The Author(s).)

The RNA-binding proteins Roquin-1/2 and Regnase-1 exert essential regulation by controlling pro-inflammatory mRNA expression to prevent autoimmune disease. More recently, inhibition of this post-transcriptional gene regulatory program has been demonstrated to enable enhanced anti-tumor responses by tumor antigen-specific CD8+ T cells. In this review, we describe the functions of these RNA-binding proteins and the phenotypes that arise in association with genetic inhibition or inactivation. We discuss how inducible inactivation of the system reprograms CD4+ and CD8+ T cell fates by changing cell metabolism, activation, differentiation or effector/memory decisions. We furthermore outline what we need to know to precisely modulate this system in order to dampen autoimmune reactions or boost the efficacy of adoptively transferred T cells or chimeric antigen receptor (CAR) T cells in cancer immunotherapies., (© The Author(s) 2022. Published by Oxford University Press on behalf of The Japanese Society for Immunology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The existence of N 6 -adenosine methylation (m 6 A) of mRNA has been known for a long time, but only recently its regulatory potential was uncovered. Current research deciphers the molecular determinants leading to the deposition of this modification and consequences for modified mRNAs. It also evaluates the importance of such modifications for specific cell types and programs. In this review, we summarize the current knowledge on m 6 A modification of mRNAs in conventional and regulatory T cells and T-cell-driven immune responses and pathology. We discuss the impact of m 6 A modification on T cell activation including cytokine and antigen receptor signaling or sensing of double-stranded RNAs (dsRNA)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

T cell antigen-receptor (TCR) signaling controls the development, activation and survival of T cells by involving several layers and numerous mechanisms of gene regulation. N 6 -methyladenosine (m 6 A) is the most prevalent messenger RNA modification affecting splicing, translation and stability of transcripts. In the present study, we describe the Wtap protein as essential for m 6 A methyltransferase complex function and reveal its crucial role in TCR signaling in mouse T cells. Wtap and m 6 A methyltransferase functions were required for the differentiation of thymocytes, control of activation-induced death of peripheral T cells and prevention of colitis by enabling gut RORγt + regulatory T cell function. Transcriptome and epitranscriptomic analyses reveal that m 6 A modification destabilizes Orai1 and Ripk1 mRNAs. Lack of post-transcriptional repression of the encoded proteins correlated with increased store-operated calcium entry activity and diminished survival of T cells with conditional genetic inactivation of Wtap. These findings uncover how m 6 A modification impacts on TCR signal transduction and determines activation and survival of T cells., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

T-cell development in the thymus is dependent on the continual colonization by bone-marrow derived progenitor cells. Once inside the thymus, progenitors undergo a series of well-defined differentiation events, including lineage commitment, somatic recombination of T-cell receptor (TCR) gene loci, and selection of clones with productively recombined yet non-autoreactive TCRs. Cell-cell interactions, cytokine signals, transcriptional as well as epigenetic programs controlling T-cell development are comparatively well-characterized. In contrast, the contribution of post-transcriptional control and its underlying mechanisms remain largely elusive. Here, we summarize recent advances in our understanding of post-transcriptional regulation of T-cell development, focussing on microRNAs (miRNAs) and RNA-binding proteins (RBPs). We highlight the current challenges, and how they can potentially be overcome with evolving sophisticated methodology to enable a thorough mechanistic understanding and decipher the regulatory networks operating in the gene expression programs of T-cell development., (Copyright © 2022. Published by Elsevier B.V.)

Extravasation of monocytes into tissue and to the site of injury is a fundamental immunological process, which requires rapid responses via post translational modifications (PTM) of proteins. Protein arginine methyltransferase 7 (PRMT7) is an epigenetic factor that has the capacity to mono-methylate histones on arginine residues. Here we show that in chronic obstructive pulmonary disease (COPD) patients, PRMT7 expression is elevated in the lung tissue and localized to the macrophages. In mouse models of COPD, lung fibrosis and skin injury, reduced expression of PRMT7 associates with decreased recruitment of monocytes to the site of injury and hence less severe symptoms. Mechanistically, activation of NF-κB/RelA in monocytes induces PRMT7 transcription and consequential mono-methylation of histones at the regulatory elements of RAP1A, which leads to increased transcription of this gene that is responsible for adhesion and migration of monocytes. Persistent monocyte-derived macrophage accumulation leads to ALOX5 over-expression and accumulation of its metabolite LTB4, which triggers expression of ACSL4 a ferroptosis promoting gene in lung epithelial cells. Conclusively, inhibition of arginine mono-methylation might offer targeted intervention in monocyte-driven inflammatory conditions that lead to extensive tissue damage if left untreated., (© 2022. The Author(s).)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Behrens and Heissmeyer.)

Roquin and Regnase-1 proteins bind and post-transcriptionally regulate proinflammatory target messenger RNAs to maintain immune homeostasis. Either the sanroque mutation in Roquin-1 or loss of Regnase-1 cause systemic lupus erythematosus-like phenotypes. Analyzing mice with T cells that lack expression of Roquin-1, its paralog Roquin-2 and Regnase-1 proteins, we detect overlapping or unique phenotypes by comparing individual and combined inactivation. These comprised spontaneous activation, metabolic reprogramming and persistence of T cells leading to autoimmunity. Here, we define an interaction surface in Roquin-1 for binding to Regnase-1 that included the sanroque residue. Mutations in Roquin-1 impairing this interaction and cooperative regulation of targets induced T follicular helper cells, germinal center B cells and autoantibody formation. These mutations also improved the functionality of tumor-specific T cells by promoting their accumulation in the tumor and reducing expression of exhaustion markers. Our data reveal the physical interaction of Roquin-1 with Regnase-1 as a hub to control self-reactivity and effector functions in immune cell therapies., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Balanced control of T cell signaling is critical for adaptive immunity and protection from autoimmunity. By combining genetically engineered mouse models, biochemical analyses and pharmacological interventions, we describe an unexpected dual role of the tumor necrosis factor receptor–associated factor 6 (TRAF6) E3 ligase as both a positive and negative regulator of mucosa-associated lymphoid tissue 1 (MALT1) paracaspase. Although MALT1-TRAF6 recruitment is indispensable for nuclear factor κB signaling in activated T cells, TRAF6 counteracts basal MALT1 protease activity in resting T cells. In mice, loss of TRAF6-mediated homeostatic suppression of MALT1 protease leads to severe autoimmune inflammation, which is completely reverted by genetic or therapeutic inactivation of MALT1 protease function. Thus, TRAF6 functions as a molecular brake for MALT1 protease in resting T cells and a signaling accelerator for MALT1 scaffolding in activated T cells, revealing that TRAF6 controls T cell activation in a switch-like manner. Our findings have important implications for development and treatment of autoimmune diseases.

Post-transcriptional gene regulation in T cells is dynamic and complex as targeted transcripts respond to various factors. This is evident for the Icos mRNA encoding an essential costimulatory receptor that is regulated by several RNA-binding proteins (RBP), including Roquin-1 and Roquin-2. Here, we identify a core RBPome of 798 mouse and 801 human T cell proteins by utilizing global RNA interactome capture (RNA-IC) and orthogonal organic phase separation (OOPS). The RBPome includes Stat1, Stat4 and Vav1 proteins suggesting unexpected functions for these transcription factors and signal transducers. Based on proximity to Roquin-1, we select ~50 RBPs for testing coregulation of Roquin-1/2 targets by induced expression in wild-type or Roquin-1/2-deficient T cells. Besides Roquin-independent contributions from Rbms1 and Cpeb4 we also show Roquin-1/2-dependent and target-specific coregulation of Icos by Celf1 and Igf2bp3. Connecting the cellular RBPome in a post-transcriptional context, we find contributions from multiple RBPs to the prototypic regulation of mRNA targets by individual trans-acting factors., (© 2021. The Author(s).)

Background: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) induced Coronavirus Disease 2019 (COVID-19) has posed a global threat to public health. The immune system is crucial in defending and eliminating the virus and infected cells. However, immune dysregulation may result in the rapid progression of COVID-19. Here, we evaluated the subsets, phenotypic and functional characteristics of natural killer (NK) and T cells in patients with COVID-19 and their associations with disease severity., Methods: Demographic and clinical data of COVID-19 patients enrolled in Wuhan Union Hospital from February 25 to February 27, 2020, were collected and analyzed. The phenotypic and functional characteristics of NK cells and T cells subsets in circulating blood and serum levels of cytokines were analyzed via flow cytometry. Then the LASSO logistic regression model was employed to predict risk factors for the severity of COVID-19., Results: The counts and percentages of NK cells, CD4 + T cells, CD8 + T cells and NKT cells were significantly reduced in patients with severe symptoms. The cytotoxic CD3 - CD56 dim CD16 + cell population significantly decreased, while the CD3 - CD56 dim CD16 - part significantly increased in severe COVID-19 patients. More importantly, elevated expression of regulatory molecules, such as CD244 and programmed death-1 (PD-1), on NK cells and T cells, as well as decreased serum cytotoxic effector molecules including perforin and granzyme A, were detected in patients with COVID-19. The serum IL-6, IL-10, and TNF-α were significantly increased in severe patients. Moreover, the CD3 - CD56 dim CD16 - cells were screened out as an influential factor in severe cases by LASSO logistic regression., Conclusions: The functional exhaustion and other subset alteration of NK and T cells may contribute to the progression and improve the prognosis of COVID-19. Surveillance of lymphocyte subsets may in the future enable early screening for signs of critical illness and understanding the pathogenesis of this disease., (Copyright © 2020 Li, Guo, Dong, Wang, Dai, Liu, Wu, Li, Zhang, Zhou, Zhang, Lin, Kang, Yu, Tian, Qin, Gui, Jiang, Fan, Heissmeyer, Sarapultsev, Wang, Luo and Hu.)

Chemical modifications are found on almost all RNAs and affect their coding and noncoding functions. The identification of m 6 A on mRNA and its important role in gene regulation stimulated the field to investigate whether additional modifications are present on mRNAs. Indeed, modifications including m 1 A, m 5 C, m 7 G, 2'-OMe, and Ψ were detected. However, since their abundances are low and tools used for their corroboration are often not well characterized, their physiological relevance remains largely elusive. Antibodies targeting modified nucleotides are often used but have limitations such as low affinity or specificity. Moreover, they are not always well characterized and due to the low abundance of the modification, particularly on mRNAs, generated data sets might resemble noise rather than specific modification patterns. Therefore, it is critical that the affinity and specificity is rigorously tested using complementary approaches. Here, we provide an experimental toolbox that allows for testing antibody performance prior to their use., (© 2020 Weichmann et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

The RNase Regnase-1 is a master RNA regulator in macrophages and T cells that degrades cellular and viral RNA upon NF-κB signaling. The roles of its family members, however, remain largely unknown. Here, we analyzed Regnase-3 -deficient mice, which develop hypertrophic lymph nodes. We used various mice with immune cell-specific deletions of Regnase-3 to demonstrate that Regnase-3 acts specifically within myeloid cells. Regnase-3 deficiency systemically increased IFN signaling, which increased the proportion of immature B and innate immune cells, and suppressed follicle and germinal center formation. Expression analysis revealed that Regnase-3 and Regnase-1 share protein degradation pathways. Unlike Regnase-1, Regnase-3 expression is high specifically in macrophages and is transcriptionally controlled by IFN signaling. Although direct targets in macrophages remain unknown, Regnase-3 can bind, degrade, and regulate mRNAs, such as Zc3h12a ( Regnase-1 ), in vitro. These data indicate that Regnase-3, like Regnase-1, is an RNase essential for immune homeostasis but has diverged as key regulator in the IFN pathway in macrophages., (© 2019 von Gamm et al.)

Post-transcriptional RNA modifications have been found to be present in a wide variety of organisms and in different types of RNA. Nucleoside modifications are interesting due to their already known roles in translation fidelity, enzyme recognition, disease progression, and RNA stability. In addition, the abundance of modified nucleosides fluctuates based on growth phase, external stress, or possibly other factors not yet explored. With modifications ever changing, a method to determine absolute quantities for multiple nucleoside modifications is required. Here, we report metabolic isotope labeling to produce isotopically labeled internal standards in bacteria and yeast. These can be used for the quantification of 26 different modified nucleosides. We explain in detail how these internal standards are produced and show their mass spectrometric characterization. We apply our internal standards and quantify the modification content of transfer RNA (tRNA) from bacteria and various eukaryotes. We can show that the origin of the internal standard has no impact on the quantification result. Furthermore, we use our internal standard for the quantification of modified nucleosides in mouse tissue messenger RNA (mRNA), where we find different modification profiles in liver and brain tissue.

The RNA-binding proteins Roquin-1 and Roquin-2 redundantly control gene expression and cell-fate decisions. Here, we show that Roquin not only interacts with stem-loop structures, but also with a linear sequence element present in about half of its targets. Comprehensive analysis of a minimal response element of the Nfkbid 3'-UTR shows that six stem-loop structures cooperate to exert robust and profound post-transcriptional regulation. Only binding of multiple Roquin proteins to several stem-loops exerts full repression, which redundantly involved deadenylation and decapping, but also translational inhibition. Globally, most Roquin targets are regulated by mRNA decay, whereas a small subset, including the Nfat5 mRNA, with more binding sites in their 3'-UTRs, are also subject to translational inhibition. These findings provide insights into how the robustness and magnitude of Roquin-mediated regulation is encoded in complex cis-elements.

T helper cell subsets orchestrate context- and pathogen-specific responses of the immune system. They mostly do so by secreting specific cytokines that attract or induce activation and differentiation of other immune or nonimmune cells. The differentiation of T helper 1 (Th1), Th2, T follicular helper, Th17, and induced regulatory T cell subsets from naive T cells depends on the activation of intracellular signal transduction cascades. These cascades originate from T cell receptor and costimulatory receptor engagement and also receive critical input from cytokine receptors that sample the cytokine milieu within secondary lymphoid organs. Signal transduction then leads to the expression of subset-specifying transcription factors that, in concert with other transcription factors, up-regulate downstream signature genes. Although regulation of transcription is important, recent research has shown that posttranscriptional and posttranslational regulation can critically shape or even determine the outcome of Th cell differentiation. In this review, we describe how specific microRNAs, long noncoding RNAs, RNA-binding proteins, and ubiquitin-modifying enzymes regulate their targets to skew cell fate decisions., (© 2018 Hoefig and Heissmeyer.)

The expression of proteins during inflammatory and immune reactions is coordinated by post-transcriptional mechanisms. A particularly strong suppression of protein expression is exerted by a conserved translational silencing element (TSE) identified in the 3' UTR of NFKBIZ mRNA, which is among the targets of the RNA-binding proteins Roquin-1/2 and MCPIP1/Regnase-1. We present evidence that in the context of the TSE MCPIP1, so far known for its endonuclease activity toward mRNAs specified by distinct stem-loop (SL) structures, also suppresses translation. Overexpression of MCPIP1 silenced translation in a TSE-dependent manner and reduced ribosome occupancy of the mRNA. Correspondingly, MCPIP1 depletion alleviated silencing and increased polysomal association of the mRNA. Translationally silenced NFKBIZ or reporter mRNAs were mostly capped, polyadenylated and ribosome associated. Furthermore, MCPIP1 silenced also cap-independent, CrPV-IRES-dependent translation. This suggests that MCPIP1 suppresses a post-initiation step. The TSE is predicted to form five SL structures. SL4 and 5 resemble target structures reported for MCPIP1 and together were sufficient for MCPIP1 binding and mRNA destabilization. Translational silencing, however, required SL1-3 in addition. Thus the NFKBIZ TSE functions as an RNA element in which sequences adjacent to the site of interaction with MCPIP1 and dispensable for accelerated mRNA degradation extend the functional repertoire of MCPIP1 to translational silencing.

The ubiquitously expressed RNA-binding proteins Roquin-1 and Roquin-2 are essential for appropriate immune cell function and postnatal survival of mice. Roquin proteins repress target mRNAs by recognizing secondary structures in their 3'-UTRs and by inducing mRNA decay. However, it is unknown if other cellular proteins contribute to target control. To identify cofactors of Roquin, we used RNA interference to screen ~1500 genes involved in RNA-binding or mRNA degradation, and identified NUFIP2 as a cofactor of Roquin-induced mRNA decay. NUFIP2 binds directly and with high affinity to Roquin, which stabilizes NUFIP2 in cells. Post-transcriptional repression of human ICOS by endogenous Roquin proteins requires two neighboring non-canonical stem-loops in the ICOS 3'-UTR. This unconventional cis-element as well as another tandem loop known to confer Roquin-mediated regulation of the Ox40 3'-UTR, are bound cooperatively by Roquin and NUFIP2. NUFIP2 therefore emerges as a cofactor that contributes to mRNA target recognition by Roquin.

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)