Identification of N6-methyladenosine (m6A) modified RNA in ILC2 of WT and Mettl3 Klrg1Cre mice

Innate lymphoid cells (ILCs) are able to directly respond to alarmin signals and produce an array of effector molecules for immune protection and tissue homeostasis. However, how posttranscriptional machinery in ILCs execute extracellular stimuli towards robust gene expression is yet to understand. Here, we reported a cell type-specific role of N6-methyladenosine (m6A) RNA methylation in ILCs. Inducible deletion of m6A methyltransferase METTL3 had little impact on ILC maintenance in the steady state or cytokine-induced ILC1 or ILC3 activation, but dramatically diminished IL-25-triggered ILC2 response. Specific deletion of Mettl3 in ILC2 significantly attenuated cell expansion, cytokine production, inter-organ migration, and anti-helminth immunity. To investigate the molecular mechanism by which m6A modification regulates ILC2 response, we subjected ILC2 of WT and Mettl3 Klrg1Cre mice to a m6A-tagged mRNA immunoprecipitation sequencing (meRIP-seq) to identify the m6A modified mRNA. We sorted ILC2 from small intestine of WT and Melttl3 Klrg1Cre mice and isolated the total RNA from the ILC2. Then we performed methylated RNA immunoprecipitation (meRIP) using anti-m6A antibody to obtain the pellet fractions which are m6A modified RNA fractions. We next constructed the libraries for pellet fractions and performed the sequencing.

固有淋巴细胞（innate lymphoid cells, ILCs）能够直接响应警报素信号，并产生一系列效应分子以参与免疫保护与组织稳态维持。然而，固有淋巴细胞内的转录后调控机制如何将胞外刺激转化为高效的基因表达程序，目前仍未阐明。本研究揭示了N6-甲基腺苷（N6-methyladenosine, m6A）RNA甲基化在固有淋巴细胞中的细胞类型特异性调控功能。研究发现，条件性敲除m6A甲基转移酶METTL3，对稳态下的固有淋巴细胞维持，或是细胞因子诱导的ILC1、ILC3活化均无显著影响，但可显著削弱IL-25触发的ILC2应答。在ILC2中特异性敲除Mettl3，可显著减弱细胞增殖、细胞因子产生、器官间迁移以及抗蠕虫免疫能力。为探究m6A修饰调控ILC2应答的分子机制，本研究对野生型（wild type, WT）与Mettl3 Klrg1Cre小鼠的ILC2进行了m6A标记的mRNA免疫沉淀测序（meRIP-seq），以鉴定携带m6A修饰的mRNA。我们从小鼠小肠中分选得到野生型与Mettl3 Klrg1Cre小鼠的ILC2，并提取其总RNA；随后使用抗m6A抗体进行甲基化RNA免疫沉淀（meRIP），获取携带m6A修饰的RNA沉淀组分；最后为该沉淀组分构建测序文库并完成测序。