RNA interference of DNA methyl-transferase 3 affects alternative splicing in the honeybee (Apis mellifera)

One of the greatest discoveries of the genomic era is environmental regulation of gene expression; DNA is not just inherited, it also is environmentally responsive. DNA cytosine methylation is an epigenetic mechanism that mediates many environmental influences on gene expression, affecting diverse aspects of organismal function and disease¹. Studies of DNA methylation from fungi, plants, and animals indicate that gene body methylation is ancient and highly conserved in eukaryotic genomes, but their roles have not been clearly defined. Regulation of alternative splicing of transcripts may have been an original function of DNA methylation, but a direct experimental test of this hypothesis has never been performed. To do this we developed a new method to administer RNA interference (RNAi) in a high-throughput and non-invasive manner, and then used this method to knock down the expression of DNA methyl-transferase 3 (dnmt3), which is required for de novo methylation. We chose the honeybee (Apis mellifera) for this test because, unlike Drosophila melanogaster, it has a fully functional methylation system and has emerged as an important model organism for studying the effects of DNA methylation on development and social behavior. In addition, methylation in honeybees is predominantly on gene bodies. Using the new TrueSight program for analyzing RNAseq data and quantifying alternative splicing, here we show that dnmt3 RNAi causes widespread and diverse changes in alternative splicing in fat tissue, affecting four different types of splicing events. These results demonstrate that one function of gene body DNA methylation is to regulate alternative splicing.