Expression profiling by high throughput sequencing
Summary
OThe N6-methyladenosine (m6A) RNA modification is widely used to alter the fate of mRNAs. Here we demonstrate that the C. elegans writer METT-10 (orthologue of mouse METTL16) deposits an m6A mark on the 3′ splice site (AG) of the SAM synthetase pre-mRNA which inhibits its proper splicing and protein production. The mechanism is triggered by a rich diet, and acts as an m6A-mediated switch to stop SAM production and regulate its homeostasis. Although the mammalian SAM synthetase pre-mRNA is not regulated via this mechanism, we show that splicing inhibition by 3′ splice site m6A is conserved in mammals. The modification functions by physically preventing the essential splicing factor U2AF35 from recognizing the 3′ splice site. We propose that use of splice site m6A is an ancient mechanism for splicing regulation.
Overall design
Abundance and distribution of m6A modification was compared between transcriptomes of the mouse and the worm by m6A-IP RNA-Seq on mixed equimolar amounts of corresponding RNAs. The effect of METT-10 on m6A distribution was analyzed by performing m6A-IP RNA-Seq on RNA isolated from wild-type and mutant METT10 worms grown either on NA22 or OP50 plates. Biological triplicates were used throuhout the study.
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