METTL3 regulates alternative splicing of cell cycle-related genes via crosstalk between mRNA mA modifications and splicing factors.

N-methyladenosine (mA) modification in RNA affects various aspects of RNA metabolism and regulates gene expression. This modification is modulated by many regulatory proteins, such as mA methyltransferases (writers), mA demethylases (erasers), and mA-binding proteins (readers). Previous studies have suggested that alterations in mA regulatory proteins induce genome-wide alternative splicing in many cancer cells. However, the functional effects and molecular mechanisms of mA-mediated alternative splicing have not been fully elucidated. To understand the consequences of this modification on RNA splicing in cancer cells, we performed RNA sequencing and analyzed alternative splicing patterns in METTL3-knockdown osteosarcoma U2OS cells. We detected 1,803 alternatively spliced genes in METTL3-knockdown cells compared to the controls and found that cell cycle-related genes were enriched in differentially spliced genes. A comparison of the published MeRIP-seq data for METTL14 with our RNA sequencing data revealed that 70-87% of alternatively spliced genes had an mA peak near 1 kb of alternative splicing sites. Among the 19 RNA-binding proteins enriched in alternative splicing sites, as revealed by motif analysis, expression of SFPQ highly correlated with METTL3 expression in 12,839 TCGA pan-cancer patients. We also found that cell cycle-related genes were enriched in alternatively spliced genes of other cell lines with METTL3 knockdown. Taken together, we suggest that METTL3 regulates mA-dependent alternative splicing, especially in cell cycle-related genes, by regulating the functions of splicing factors such as SFPQ.