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. Overall design: 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).

N6-甲基腺苷酸（N6-methyladenosine，m6A）是成熟信使RNA（mRNA）中最为丰富的转录后甲基化修饰，在包括发育进程与癌症发生发展在内的多种生物学功能中发挥关键调控作用。m6A由METTL3与METTL14组成的甲基转移酶复合物共转录沉积，并可被去甲基化酶FTO或ALKBH5催化移除。m6A在成熟mRNA的3'非翻译区（3' UTR）以及终止密码子附近呈现高度富集特征。然而，造就这一分布模式的分子机制至今仍不明晰。本研究证实，外显子连接复合物（Exon Junction Complex，EJC）通过包装mRNA来塑造其m6A修饰图谱。我们首先验证了去甲基化酶在剪接过程中移除m6A的可能性，实验结果显示ALKBH5敲降对m6A水平仅存在微弱影响，因此我们排除了去甲基化调控模型。随后我们提出假说：剪接因子会抑制内部外显子的甲基化过程，但不作用于3' UTR区域。敲降外显子连接复合物的核心组分EIF4A3后，成熟mRNA中的m6A水平显著升高，这些高甲基化位点富集于短的内部外显子中。进一步研究发现，EIF4A3敲降可增强METTL3与mRNA的结合亲和力，进而促进m6A修饰的沉积。综上，本研究结果表明，EIF4A3通过阻断METTL3的结合，抑制mRNA内部短外显子的甲基化过程。上述发现揭示了剪接过程中形成的RNA包装结构可作为调控因子，塑造mRNA的修饰图谱。整体实验设计：对HeLa细胞及其敲降衍生物（siALKBH5与siEIF4A3）的meRIP-seq数据开展比较性m6A修饰谱分析，并对eCLIP-seq数据进行METTL3结合谱分析。