RNA-seq of arsenite-induced stress granule transcriptomes in control and Mettl3- and m6A-depleted mouse embryonic fibroblasts (MEFs)

N6-methyladenosine (m6A) is the most abundant nucleotide modification found on mRNA. m6A has been previously shown to facilitate recruitment of mRNAs to stress granules due to interactions between m6A and the YTHDF proteins. Although protein-RNA interactions are known to contribute to the recruitment of mRNAs to stress granules, the predominant driver of mRNA recruitment to stress granules is thought to be RNA-RNA interactions between long RNAs. Here, we show that m6A contributes significantly to the length-dependent effect of mRNA enrichment in stress granules and that upon depletion of METTL3, the enzyme responsible for adding m6A to nascent transcripts, the nature of the stress granule transcriptome is fundamentally altered by greatly reducing the propensity of long mRNAs to enter stress granules. This reveals that not only does m6A enhance the phase separation of mRNAs, but that it is a driving force underpinning length-dependent enrichment of mRNAs in stress granules. Overall design: RNA-seq was performed on control and METTL3- and m6A-depleted E13.5 mouse embryonic fibroblasts. Total mRNA samples were harvested under standard conditions. Stress granule-enriched mRNA fractions were isolated following arsenite stress (500 uM, 30 min) and stress granule enrichment via centrifugation. Each condition has two biological replicates.