METTL3 regulates alternative splicing of cell cycle-related genes via crosstalk between mRNA m(6)A modifications and splicing factors

Abstract

N-6-methyladenosine (m(6)A) modification in RNA affects various aspects of RNA metabolism and regulates gene expression. This modification is modulated by many regulatory proteins, such as m(6)A methyltransferases (writ-ers), m(6)A demethylases (erasers), and m(6)A-binding proteins (readers). Previous studies have suggested that al-terations in m6A regulatory proteins induce genome-wide alternative splicing in many cancer cells. However, the functional effects and molecular mechanisms of m6A-mediated alternative splicing have not been fully elucidated. To understand the consequences of this modification on RNA splicing in cancer cells, we performed RNA sequenc-ing 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 m6A 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 m(6)A-dependent alternative splicing, espe-cially in cell cycle-related genes, by regulating the functions of splicing factors such as SFPQ.