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

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 ROR?t+ 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. Overall design: Examination of transcriptome in WT or Wtap KO CD4+ T cells. m6A-CLIP or YTHDF2-iCLIP experiments were performed on polyA+ RNA from mouse CD4+ cells with 3 or 2 biological replicates, respectively.

T细胞抗原受体（T cell antigen receptor, TCR）信号通路通过多层级的基因调控环节与多种调控机制，调控T细胞的发育、活化与存活。N6-甲基腺苷酸（N6-methyladenosine, m6A）是分布最为广泛的信使RNA（mRNA）修饰形式，可影响转录本的剪接、翻译及稳定性。本研究证实Wtap蛋白是m6A甲基转移酶复合物发挥功能的必需因子，并揭示其在TCR信号通路中的关键调控作用。Wtap及m6A甲基转移酶的功能对于胸腺细胞分化、外周T细胞激活诱导性死亡的调控，以及通过维持肠道RORγt阳性调节性T细胞功能以预防结肠炎均至关重要。转录组与表观转录组学分析显示，m6A修饰可降低Orai1与Ripk1 mRNA的稳定性。编码蛋白的转录后抑制缺失，会伴随钙池操纵性钙内流（store-operated calcium entry, SOCE）活性增强以及T细胞存活能力下降。本研究阐明了m6A修饰调控TCR信号转导的具体机制，并明确其对T细胞活化与存活的决定性作用。整体实验设计：对野生型（wild type, WT）或Wtap敲除（knockout, KO）的CD4阳性T细胞开展转录组分析。分别以3次和2次生物学重复，对小鼠CD4阳性细胞的聚腺苷酸化RNA（polyA+ RNA）进行m6A-CLIP或YTHDF2-iCLIP实验。