Exon junction complex shapes m6A epitranscriptome during splicing

N6-methyladenosine (m6A) is the most abundant post-transcriptional methylation of mature mRNA and plays a crucial regulatory role in various biological functions, involving development and cancer progression. m6A is deposited by methyltransferase complex composed of METTL3 and METTL14 cotranscriptionally, and removed by demethylase FTO or ALKBH5. m6A is highly enriched within the 3’ UTRs and in the vicinity of the stop codon of mature mRNA. However, the mechanism that causes this distribution pattern is still enigmatic. Here, we show that EJC packages mRNAs to shape mRNA m6A landscape. We first tested the possibility that demethylase removes m6A during spicing and found that ALKBH5 depletion had a minor effect on m6A levels. Thus, we ruled out the demethylation model. We then hypothesized that splicing factors inhibit the methylation process of internal exons, but not the 3’ UTR. Knockdown of EIF4A3, a core component of the exon junction complex, significantly increased m6A levels in mature mRNAs. The hypermethylated sites are enriched in short internal exons. Furthermore, EIF4A3 depletion upregulates METTL3 binding affinity to mRNA to deposit m6As. In conclusion, Our results demonstrate that EIF4A3 blocks METTL3 binding and methylating internal short exons of mRNA. These findings shed light on the fact that RNA packaging formed during splicing acts as a regulator that shapes mRNA modifications. Comparative m6A modification profiling analysis of meRIP-seq data and METTL3 binding profiling analysis of eCLIP-seq data for HeLa cells and its KD derivatives (siALKBH5 and siEIF4A3).