As the most abundant eukaryotic mRNA modification, N6-methyladenosine (m6A) is crucial in regulating multiple biological processes. This methylation is regulated by methyltransferases and demethylases. However, the regulatory role and mode of action of m6A demethylases in fungi remain poorly understood. Here, we show that CpALKBH is a demethylase in Cryphonectria parasitica that removes m6A modification from single-stranded RNA in vitro. The deletion of CpALKBH resulted in a significant elevation in the m6A methylation level and a decrease in the growth rate, sporulation, and virulence of the fungus. Additionally, CpZap1 was identified as a downstream target of CpALKBH demethylase based on RNA sequencing. We confirmed that CpALKBH demethylase regulates CpZap1 mRNA stability in an m6A dependent manner. Furthermore, through MazF assay, CpZap1 methylation at position 1935A was found to be regulated by both CpALKBH demethylase and CpMTA1 methyltransferase.
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