RNA modifications on Adenosine Regulate Macrophage Function

Macrophages are important innate immune cells that respond rapidly to inflammatory cues and fulfill diverse functions in the activation, execution and resolution of immune and inflammatory responses. Currently, there is no comprehensive understanding of the combinatorial effect of mRNA modifications in a single cell type, the consequences for mRNA processing and metabolism, and how these changes affect cell physiology. We use the mouse macrophage line RAW264.7 (RAW) to systematically address the combinatorial effect of two major mRNA modifications during macrophage stimulation: adenosine to inosine deamination (A-to-I), and adenosine N6 methylation (m6A). At the functional level we found that both METTL3 (the m6A writer) and ADAR1 (the adenosine deaminase) are required for full activation of RAW macrophages (as determined by expression of surface markers) and for optimal phagocytosis (a key macrophage function). Using both short read and single molecule sequencing on RAW cells with genetic deletions of the m6A writer METTL3 and the A deaminase ADAR1, we found that loss of m6A affected A-to-I levels while m6A levels remained relatively stable. A detailed investigation of potential interactions between m6A and A-to-I at the single transcript level is ongoing.