The function of Wtap in N6-adenosine methylation of mRNAs controls T cell receptor signaling and survival of T cells.

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. N6-methyladenosine (m6A) 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 m6A methyltransferase complex function and reveal its crucial role in TCR signaling in mouse T cells. Wtap and m6A 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 m6A 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 m6A modification impacts on TCR signal transduction and determines activation and survival of T cells.

Validation strategies for antibodies targeting modified ribonucleotides.

Chemical modifications are found on almost all RNAs and affect their coding and noncoding functions. The identification of m6A on mRNA and its important role in gene regulation stimulated the field to investigate whether additional modifications are present on mRNAs. Indeed, modifications including m1A, m5C, m7G, 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.

Essential functions of the CNOT7/8 catalytic subunits of the CCR4-NOT complex in mRNA regulation and cell viability.

Shortening of mRNA poly(A) tails (deadenylation) to trigger their decay is mediated mainly by the CCR4-NOT deadenylase complex. While four catalytic subunits (CNOT6, 6L 7, and 8) have been identified in the mammalian CCR4-NOT complex, their individual biological roles are not fully understood. In this study, we addressed the contribution of CNOT7/8 to viability of primary mouse embryonic fibroblasts (MEFs). We found that MEFs lacking CNOT7/8 expression [Cnot7/8-double knockout (dKO) MEFs] undergo cell death, whereas MEFs lacking CNOT6/6L expression (Cnot6/6l-dKO MEFs) remain viable. Co-immunoprecipitation analyses showed that CNOT6/6L are also absent from the CCR4-NOT complex in Cnot7/8-dKO MEFs. In contrast, either CNOT7 or CNOT8 still interacts with other subunits in the CCR4-NOT complex in Cnot6/6l-dKO MEFs. Exogenous expression of a CNOT7 mutant lacking catalytic activity in Cnot7/8-dKO MEFs cannot recover cell viability, even though CNOT6/6L exists to some extent in the CCR4-NOT complex, confirming that CNOT7/8 is essential for viability. Bulk poly(A) tail analysis revealed that mRNAs with longer poly(A) tails are more numerous in Cnot7/8-dKO MEFs than in Cnot6/6l-dKO MEFs. Consistent with elongated poly(A) tails, more mRNAs are upregulated and stabilized in Cnot7/8-dKO MEFs than in Cnot6/6l-dKO MEFs. Importantly, Cnot6/6l-dKO mice are viable and grow normally to adulthood. Taken together, the CNOT7/8 catalytic subunits are essential for deadenylation, which is necessary to maintain cell viability, whereas CNOT6/6L are not.

The thymocyte-specific RNA-binding protein Arpp21 provides TCR repertoire diversity by binding to the 3'-UTR and promoting Rag1 mRNA expression.

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 Ca2+ 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.

Critical functions of N6-adenosine methylation of mRNAs in T cells.

The existence of N6-adenosine methylation (m6A) 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 m6A modification of mRNAs in conventional and regulatory T cells and T-cell-driven immune responses and pathology. We discuss the impact of m6A modification on T cell activation including cytokine and antigen receptor signaling or sensing of double-stranded RNAs (dsRNA).

The mRNA methyltransferase Mettl3 modulates cytokine mRNA stability and limits functional responses in mast cells.

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 N6-methyladenosine (m6A) 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 m6A 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 m6A sites in the Il13 3'-untranslated region. Overall, we reveal that the m6A machinery is essential in mast cells to sustain growth and to restrain inflammatory responses.

Defining the RBPome of primary T helper cells to elucidate higher-order Roquin-mediated mRNA regulation.

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.

Potent and broad anticancer activities of leaf extracts from Melia azedarach L. of the subtropical Okinawa islands.

Plant extracts have been traditionally used for various therapeutic applications. By conducting an initial screening of several subtropical plants, in this study, we evaluated the anticancer activities of Melia azedarach L. The extract from Melia azedarach L. leaves (MLE) show high cytotoxic effects on cancer cells and in vivo mouse and dog tumor models. During the initial screening, MLE showed strong antiproliferative activity against HT-29 colon, A549 lung, and MKN1 gastric cancer cells. In subsequent tests, using 39 human tumor cell lines, we confirmed the potent anticancer activities of MLE. The anticancer activity of MLE was also confirmed in vivo. MLE markedly inhibited the growth of transplanted gastric MKN1 cancer xenografts in mice. To elucidate the mechanism underlying the anticancer effects of MLE, MLE-treated MKN1 cells were observed using an electron microscope; MLE treatment induced autophagy. Furthermore, western blot analysis of proteins in lysates of MLE-treated cells revealed induction of light chain 3 (LC3)-II autophagosomal proteins. Thus, MLE appeared to suppress MKN1 cell proliferation by inducing autophagy. In addition, in the mouse macrophage cell line J774A.1, MLE treatment induced TNF-α production, which might play a role in tumor growth suppression in vivo. We also performed a preclinical evaluation of MLE treatment on dogs with various cancers in veterinary hospitals. Dogs with various types of cancers showed a mean recovery of 76% when treated with MLE. Finally, we tried to identify the active substances present in MLE. All the active fractions obtained by reverse-phase chromatography contained azedarachin B-related moieties, such as 3-deacetyl-12-hydroxy-amoorastatin, 12-hydroxy-amoorastatin, and 12-hydroxyamoorastaton. In conclusion, MLE contains substances with promising anticancer effects, suggesting their future use as safe and effective anticancer agents.

The CCR4-NOT deadenylase complex safeguards thymic positive selection by down-regulating aberrant pro-apoptotic gene expression.

A repertoire of T cells with diverse antigen receptors is selected in the thymus. However, detailed mechanisms underlying this thymic positive selection are not clear. Here we show that the CCR4-NOT complex limits expression of specific genes through deadenylation of mRNA poly(A) tails, enabling positive selection. Specifically, the CCR4-NOT complex is up-regulated in thymocytes before initiation of positive selection, where in turn, it inhibits up-regulation of pro-apoptotic Bbc3 and Dab2ip. Elimination of the CCR4-NOT complex permits up-regulation of Bbc3 during a later stage of positive selection, inducing thymocyte apoptosis. In addition, CCR4-NOT elimination up-regulates Dab2ip at an early stage of positive selection. Thus, CCR4-NOT might control thymocyte survival during two-distinct stages of positive selection by suppressing expression levels of pro-apoptotic molecules. Taken together, we propose a link between CCR4-NOT-mediated mRNA decay and T cell selection in the thymus.

Production and Application of Stable Isotope-Labeled Internal Standards for RNA Modification Analysis.

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.

Tropomodulin 1 expression driven by NF-κB enhances breast cancer growth.

Triple-negative breast cancers (TNBC), which include the basal-like and claudin-low disease subtypes, are aggressive malignancies for which effective therapeutic targets are lacking. NF-κB activation has an established role in breast malignancy, and it is higher in TNBC than other breast cancer subtypes. On this basis, we hypothesized that proteins derived from NF-κB target genes might be molecular targets for TNBC therapy. In this study, we conducted a microarray-based screen for novel NF-κB-inducible proteins as candidate therapeutic targets, identifying tropomodulin 1 (TMOD1) as a lead candidate. TMOD1 expression was regulated directly by NF-κB and was significantly higher in TNBC than other breast cancer subtypes. TMOD1 elevation is associated with enhanced tumor growth in a mouse tumor xenograft model and in a 3D type I collagen culture. TMOD1-dependent tumor growth was correlated with MMP13 induction, which was mediated by TMOD1-dependent accumulation of ß-catenin. Overall, our study highlighted a novel TMOD1-mediated link between NF-κB activation and MMP13 induction, which accounts in part for the NF-κB-dependent malignant phenotype of TNBC.

NF-κB non-cell-autonomously regulates cancer stem cell populations in the basal-like breast cancer subtype.

Patients with triple-negative breast cancer display the highest rates of early relapse of all patients with breast cancer. The basal-like subtype, a subgroup of triple-negative breast cancer, exhibits high levels of constitutively active NF-κB signalling. Here we show that NF-κB activation, induced by inflammatory cytokines or by epigenetically dysregulated NIK expression, cell-autonomously upregulates JAG1 expression in non-cancer stem cells. This upregulation stimulates NOTCH signalling in cancer stem cells in trans, leading to an expansion of cancer stem cell populations. Among breast cancers, the NF-κB-dependent induction of JAG1 and the NOTCH-dependent expansion of the cancer stem cell population occur only in the basal-like subtype. Collectively, our results indicate that NF-κB has a non-cell-autonomous role in regulating cancer stem cell populations by forming intratumoural microenvironments composed of JAG1-expressing non-cancer stem cells with a basal-like subtype.