Pooled CRISPR Screening Identifies m6A as a Positive Regulator of Macrophage Activation [RNA-seq]

The involvement of m6A modification in macrophage activation has been validated in our study that the expression of TNF-α in Mettl3-depleted Raw 264.7 cells stimulated with LPS were markedly reduced in comparison to control cells. To further explore the biological effects of m6A deficiency macrophages, we performed RNA sequencing analysis of Mettl3-KO and WT control Raw 264.7 cells upon LPS treatment. The GO enrichment analysis documented that the downregulated transcripts in Mettl3-KO Raw 264.7 cells were enriched in innate immune response related to defense and external stimulus. Notably, transcripts of the downstream components of the TLR4 signaling pathway, such as proinflammatory cytokines (Tnf-α, Il-6, Il-1β, Il-18,and Il-23) and co-stimulation molecules (Cd86), were downregulated in Mettl3-deficient cells, suggesting that METTL3 has a critical function in controlling the innate immune response of Raw 264.7 macrophages. RNA samples from Mettl3-KO and WT control Raw 264.7 cells upon LPS treatment were collected, and then RNA purification, reverse transcription, library construction and sequencing were performed according to the manufacturer's instructions (Illumina).