The mRNA methyltransferase Mettl3 modulates cytokine mRNA stability and limits functional responses in mast cells [NanoString]

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 revealed that the m6A mRNA methyltransferase complex modulates mast cell proliferation and survival. Depletion of the catalytic component Mettl3 exacerbated effector functions in response to IgE and antigen complexes, both in vitro and in vivo. Mechanistically, deletion of Mettl3 or Mettl14 led 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 found that it was methylated in activated mast cells, and that Mettl3 affected its transcript stability in an enzymatic activity-dependent manner, requiring consensus m6A sites in the Il13 3’-untranslated region. Overall, we revealed that the m6A machinery is essential in mast cells to sustain growth and to restrain inflammatory responses. 3 sets of in vitro bone marrow derived mast cells were CRISPR/Cas9 modified to knockout Mettl3 or cKit, as a control, and insert a zsGreen reporter gene at the knockout target site. Cells were sorted for high zsGreen expression and were either left resting or were stimulated for 4h with IgE and antigen complexes.