METTL14-Mediated N6-Methyladenosine Exacerbates Acute Lung Injury via USP30/NCOA4-Dependent Ferroptosis.

Ischemia/reperfusion (I/R)-induced acute lung injury (ALI) is a severe clinical syndrome characterized by alveolar epithelial damage, oxidative stress, and acute respiratory failure, but the epitranscriptomic mechanisms linking N6-methyladenosine (m6A) modification to ferroptosis remain unclear. Clinical profiling of end-stage acute respiratory distress syndrome (ARDS) patients showed hyperinflammation, reflected by elevated C-reactive protein and procalcitonin, coagulopathy with abnormal D-dimer levels, and severely impaired gas exchange. Reanalysis of a public murine single-cell RNA sequencing dataset revealed enrichment of RNA methylation-, ubiquitination-, and ferroptosis-related programs in injured lung epithelial cells. Integrated methylated RNA immunoprecipitation sequencing and RNA sequencing of I/R-injured mouse lungs further identified methyltransferase-like 14 (METTL14) and YTH N6-methyladenosine RNA-binding protein 1 (YTHDF1) as candidate m6A regulators. Human ARDS tissues and mouse I/R lungs confirmed increased METTL14, ubiquitin-specific peptidase 30 (USP30), and nuclear receptor coactivator 4 (NCOA4) expression. Mechanistically, METTL14-mediated m6A modification enhanced USP30 expression through YTHDF1 recognition, whereas USP30 stabilized NCOA4 by reducing K48-linked polyubiquitination. Stabilized NCOA4 promoted ferritinophagy, Fe2 + accumulation, lipid peroxidation, and epithelial ferroptosis. These findings identify a METTL14/YTHDF1-USP30-NCOA4 axis as a potential therapeutic target for ferroptosis-driven I/R-induced ALI.