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The function of Wtap in N6-adenosine methylation of mRNAs controls T cell receptor signaling and survival of T cells [RNA-seq]

ABSTRACT: 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 mRNA modification affecting splicing, translation and stability of transcripts. Here, we describe the Wtap protein as essential for m6A methyltransferase complex function and reveal its crucial role in TCR signaling. 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 (SOCE) activity and diminished survival of T cells. These findings uncover how m6A modification impacts on TCR signal transduction and determines activation and survival of T cells.

ORGANISM(S): Mus musculus

PROVIDER: GSE188814 | GEO | 2022/06/03

REPOSITORIES: GEO

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The function of Wtap in N6-adenosine methylation of mRNAs controls T cell receptor signaling and survival of T cells [m6A-CLIP]

Project description: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 mRNA modification affecting splicing, translation and stability of transcripts. Here, we describe the Wtap protein as essential for m6A methyltransferase complex function and reveal its crucial role in TCR signaling. 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 (SOCE) activity and diminished survival of T cells. These findings uncover how m6A modification impacts on TCR signal transduction and determines activation and survival of T cells.

2022-06-03 | GSE188853 | GEO

The function of Wtap in N6-adenosine methylation of mRNAs controls T cell receptor signaling and survival of T cells [YTHDF2-iCLIP]

2022-06-03 | GSE188846 | GEO

Profiling of N6-methyladenosine in HepG2 cells: SETD2, METTL3, METTL14 or WTAP knockdown and control cells

Project description:SETD2 is the specific methyltransferase of H3K36me3, while METTL3, METTL14 and WTAP are the components of m6A methyltransferase complex. To understand the global effect of H3K36me3 on m6A modification, we compared the m6A profiling in SETD2 and METTL3, METTL14 or WTAP knockdown HepG2 cells, and found depletion of H3K36me3 by SETD2 silencing globally reduced m6A in human transcriptome. What’s more, most of the SETD2-dependent hypomethylation sites also responded to knockdown of METTL3, METTL14, or WTAP.

2019-02-21 | GSE110320 | GEO

WTAP-mediated m6A modification profiling in human nasopharyngeal carcinoma cell lines

Project description:N(6)-methyladenosine (m6A) plays an important role in the tumorigenesis and progression of cancers. However, the clinical significance of m6A and their regulatory mechanisms in nasopharyngeal carcinogenesis (NPC) remain largely unknown. In this study, we used the microarray analysis to study WTAP-mediated m6A modification profiles in human nasopharyngeal carcinoma cell line, HONE-1, by comparing 3 pairs of samples with or without WTAP knockdown.

2022-06-15 | GSE160644 | GEO

Arabidopsis Messenger RNA N6-methyladenosine Demethylation Regulates Plant Development

Project description:N6-methyladenosine (m6A) is the most abundant internal modification in the messenger RNA (mRNA) of all higher eukaryotes. This modification has been shown to be reversible in mammals; it is installed by a methyltransferase heterodimer complex of METTL3 and METTL14 bound with WTAP, and reversed by iron(II)- and Î±-ketoglutarate-dependent demethylases FTO and ALKBH5. This modification exhibits significant functional roles in various biological processes. The m6A modification as a RNA mark is recognized by reader proteins, such as YTH domain family proteins and HNRNPA2B1; m6A can also act as a structure switch to affect RNA-protein interactions for biological regulation. In Arabidopsis thaliana, the methyltransferase subunit MTA (the plant orthologue of human METTL3, encoded by At4g10760) was well characterized and FIP37 (the plant orthologue of human WTAP) was first identified as the interacting partner of MTA. Here we report the discovery and characterization of reversible m6A methylation mediated by AtALKBH10B (encoded by At4g02940) in A. thaliana, and noticeable roles of this RNA demethylase in affecting plant development and floral transition. Our findings reveal potential broad functions of reversible mRNA methylation in plants. m6A peaks were identified from wild type Columbia-0 and atalkbh10b-1 mutant in two biological replicates

2016-03-24 | E-GEOD-79523 | biostudies-arrayexpress

Identification and Characterization of the Mammalian Nuclear RNA N6-Adenosine Methyltransferase Core Complex

Project description:N6-methyladenosine (m6A) is the most prevalent internal modification found in mammalian messenger and non-coding RNAs. The discoveries of functionally significant demethylases that reverse this methylation as well as the recently revealed m6A distributions in mammalian transcriptomes strongly indicate regulatory functions of this modification. Here we report the identification and characterization of the mammalian nuclear RNA N6-adenosine methyltransferase core (RNMTC) complex. Besides METTL3, a methyltransferase which was the only known component of RNMTC in the past, we discovered that a previously uncharacterized methyltransferase, METTL14, exhibits a N6-adenosine methyltransferase activity higher than METTL3. Together with WTAP, the third component that dramatically affects the cellular m6A level, these three proteins form the core complex that orchestrates m6A deposition on mammalian nuclear RNA. Biochemistry assays, imaging experiments, as well as transcriptome-wide analyses of the binding sites and their effects on m6A methylation support methylation function and reveal new insights of RNMTC. PAR-CLIP and m6A-seq in HeLa cells

2013-11-29 | E-GEOD-46705 | biostudies-arrayexpress

Differential regulation of membrane potential during store-operated calcium entry in neutrophil subsets

Project description:Calcium signals are initiated in immune cells by the process of store-operated calcium entry (SOCE), where receptor activation triggers transient calcium release from the endoplasmic reticulum, followed by opening of plasma membrane calcium-release activated calcium (CRAC) channels. ORAI1, ORAI2 and ORAI3 are known to comprise the CRAC channel, however the contributions of individual isoforms to neutrophil function is not well understood. Here we show that loss of ORAI1 partially decreases calcium influx while loss of both ORAI1 and ORAI2 completely abolishes store-operated calcium entry. In other immune cell types, loss of ORAI2 enhances SOCE. In contrast, we find that ORAI2-deficient neutrophils display decreased calcium influx, which is correlated with measurable differences in regulation of neutrophil membrane potential via KCa3.1. Decreased SOCE in ORAI1-, ORAI2- and ORAI1/2-deficient neutrophils impairs multiple neutrophil functions including phagocytosis, degranulation, leukotriene and ROS production, rendering ORAI1/2-deficient mice highly susceptible to staphylococcal infection. This study demonstrates that ORAI1 and ORAI2 are the primary components of the neutrophil CRAC channel and identifies novel subpopulations of neutrophils where cell membrane potential functions as a rheostat to modulate the SOCE response. These findings have implications for new mechanisms that modulate neutrophil function during infection, acute and chronic inflammatory conditions, and cancer.

2020-09-01 | GSE157200 | GEO

Characterization of A Novel CRAC Inhibitor that Potently Blocks Human T cell Activation and Effector Functions

Project description:Store operated calcium entry (SOCE) downstream of T cell receptor (TCR) activation in T lymphocytes has been shown to be mediated mainly through the Calcium Release Activated Calcium (CRAC) channel. Here, we compared the effects of a novel, potent and selective CRAC inhibitor, 2,6-Difluoro-N-{5-[4-methyl-1-(5-methyl-thiazol-2-yl)-1,2,5,6-tetrahydro-pyridin-3-yl]-pyrazin-2-yl}-benzamide (RO2959), on T cell effector functions with that of a previously reported CRAC channel inhibitor, YM-58483, and a calcineurin inhibitor Cyclosporin A (CsA). Using both electrophysiological and calcium-based fluorescence measurements, we showed that RO2959 is a potent SOCE inhibitor that blocked an IP3-dependent current in CRAC-expressing RBL-2H3 cells and CHO cells stably expressing human Orai1 and Stim1, as well as SOCE in human primary CD4+ T cells triggered by either TCR stimulation or thapsigargin treatment. Furthermore, we demonstrated that RO2959 completely inhibited cytokine production as well as T cell proliferation mediated by TCR stimulation or MLR (Mixed Lymphocyte Reaction). Lastly, we showed by gene expression array analysis that RO2959 potently blocked TCR triggered gene expression and T cell functional pathways similar to CsA and FK506. Thus, both from a functional and transcriptional level, our data provide evidence that RO2959 is a novel and selective CRAC inhibitor that potently inhibits human T cell functions. PBMC from healthy donors (n=4) were stimulated with anti-CD3/CD28 in the presence or absence of CRAC inhibitor, CsA or FK506 for 24 hrs

2013-01-31 | E-GEOD-43210 | biostudies-arrayexpress

High-resolution mapping reveals a conserved, widespread, dynamic meiotically regulated mRNA methylation program [Mm]

Project description:N6-methyladenosine (m6A) is a common modification of mRNA, with potential roles in fine-tuning the RNA life cycle, but little is known about the pathways regulating this process and its physiological role. Here, we used mass-spectrometry to identify a dense network of proteins physically interacting with METTL3, a core component of the methyltransferase complex, and show that two of them, WTAP and KIAA1429, are required for methylation. Combining high resolution m6A-Seq with knockdown of WTAP allowed us to define accurate maps, at near single-nucleotide resolution, of sites of mRNA methylation across four dynamic programs in human and mouse, including development, differentiation, reprogramming and immune response. Internal WTAP-dependent methylation sites were largely static across the different surveyed conditions and present in the majority of mRNAs. However, methylations were found at much lower levels within highly expressed mRNAs, and methylation is inversely correlated with mRNA stability, consistent with a role in establishing an overall basal, cell-type invariant, distribution of degradation rates. In addition, we identify thousands of WTAP-independent methylation sites at transcription initiation sites, forming part of the mRNA cap structure. We show that the methylations occur at the first transcribed nucleotide, and find that thousands of transcripts are present in different isoforms differing in the methylation state of the first transcribed nucleotide, a previously unappreciated complexity of the transcriptome. Together, our data sheds new light on the proteomic and transcriptional underpinnings of this epitranscriptomic modification in mammals. Examination of m6A methylation across different knockdowns using shRNAs in mouse embryonic fibroblasts, in embyronic and adult brains, and in dendritic cell stimulated with LPS.

2014-07-18 | E-GEOD-55575 | biostudies-arrayexpress

The effects of WTAP on m6A mRNA modification pancreatic islets

Project description:Through m6A mRNA-profiling, we aim to characterize the m6A mRNA changes in pancreatic islets between Wtapflox/flox and Wtap-βKO mice.

2023-01-31 | GSE215360 | GEO

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