The splicing factor U2AF65B regulates cytosine methylation through interacting with DEFECTIVE IN MERISTEM SILENCING 3...

U2AF65B is one of the splicing factors that are involved in the recognition of the 3' splicing site and it plays an important role in plant development and stress response through its mRNA splicing function. However, it is not clear whether U2AF65B regulates gene expression in a splicing-independent manner. Through mutant screening and map-based cloning, protein-protein interaction, transcriptomic sequencing, whole-genome bisulfite sequencing and chromatin immunoprecipitation analysis, we investigated the function of U2AF65B in gene silencing in Arabidopsis thaliana. We found in the u2af65b mutant that the exogenous transgene 35S::HYG is activated in expression with decreased DNA methylation on the 35S core-promoter compared with that in the wild-type. Loss of U2AF65B function also globally decreased the methylation of CG, CHG and CHH with a profound effect on CHH methylation in transposons and intergenic sequences. Among the hypomethylated non-CG cytosines in u2af65b, nearly half of them are also hypomethylated in the dms3 mutant. Interestingly, U2AF65B interacts with the RNA-directed DNA methylation (RdDM) pathway component DMS3, and loss of U2AF65B function significantly decreased the enrichment of DMS3 on the targets, including the 35S::HYG transgene and endogenous RdDM loci. Our findings suggest that U2AF65B is a crucial player in RdDM-mediated DNA methylation, partially through promoting the RdDM pathway by interacting with and recruiting DMS3 to the target sequences.