Identification of N6-methyladenosine (m6A) modified RNA in activated ILC1

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 ILC response, we subjected IL-12 plus IL-18-activated ILC1 to a m6A-tagged mRNA immunoprecipitation sequencing (meRIP-seq) to identify the m6A modified mRNA. Overall design: We sorted IL-12 plus IL-18-induced ILC1 that were activated ILC1 in liver of C57BL/6 mice and isolated the total RNA from the ILC1. Then we performed methylated RNA immunoprecipitation (meRIP) using anti-m6A antibody to obtain the unbound and pellet fractions. The unbound fraction is non-m6A modified RNA fraction while the pellet fraction is m6A modified RNA fraction. We next constructed the libraries for unbound and pellet fractions and performed the sequencing.