Endotoxin-induced m6A RNA methylation landscape in lung endothelial cells: role of METTL3 in regulating inflammation and injury during acute lung injury.

Acute Lung Injury (ALI) involves diffuse alveolar damage, neutrophil infiltration, and pulmonary edema, with unacceptable mortality. Bacterial lipopolysaccharide (LPS) activates inflammatory pathways in ALI, which are then regulated by transcriptional and post-transcriptional pathways to affect gene expression. RNA methylation, N6-methyladenosine, is the main m6A mRNA modification that controls the expression of various genes in different environments. There are very few facts about LPS's effect on m6A RNA methylation. This study will explore the m6A RNA methylation landscape in lung endothelial cells (ECs) to understand its role in lung inflammation. In this study, lung endothelial cells were treated with LPS, and the dynamics of mRNA m6A methylation were examined through m6A-methylated RNA sequencing. RNA abundance was measured with RNA-seq, and global protein expression and m6A-binding proteins were identified using mass spectrometry (MS). Following LPS treatment, global m6A methylation levels increased along with the upregulation and nuclear translocation of METTL3 protein, while demethylase activity remained unchanged. METTL3 drove LPS-induced m6A methylation and endothelial injury, as shown by selective METTL3 siRNA and the inhibitor STM2457. MeRIP-seq analyses revealed increased m6A sites near the 5' UTR in LPS-treated cells, with m6A methylation correlating positively with gene expression. The metabolic and apoptosis pathways were shown to be more enriched in different types of methylated exons. METTL3-mediated m6A methylation targeted inflammatory genes, enhancing protein expression in chemokine signaling and MAPK pathways. STM2457 effectively mitigated LPS- or CLP-induced experimental ALI. According to this paper, LPS-mediated m6A RNA methylation is described in terms of genomic structure. Modulation of m6A methylation exerts influence over LPS-mediated endothelial gene expression and the ensuing inflammatory response.