Fragile X mental retardation protein modulates the stability of its m6A-marked messenger RNA targets

N6-methyladenosine (m6A) is the most prevalent internal modification of mammalian messenger RNAs (mRNAs) and long non-coding RNAs. The biological functions of this reversible RNA modification can be interpreted by cytoplasmic and nuclear "m6A reader" proteins to fine-tune gene expression, such as mRNA degradation and translation initiation. Here we profiled transcriptome-wide m6A sites in adult mouse cerebral cortex, underscoring that m6A is a widespread epitranscriptomic modification in brain. Interestingly, the mRNA targets of fragile X mental retardation protein (FMRP), a selective RNA-binding protein, are enriched for m6A marks. Loss of functional FMRP leads to Fragile X syndrome (FXS), the most common inherited form of intellectual disability. Transcriptome-wide gene expression profiling identified 2,035 genes differentially expressed in the absence of FMRP in cortex, and 92.5% of 174 downregulated FMRP targets are marked by m6A. Biochemical analyses indicate that FMRP binds to the m6A sites of its mRNA targets and interacts with m6A reader YTHDF2 in an RNA-independent manner. FMRP maintains the stability of its mRNA targets while YTHDF2 promotes the degradation of these mRNAs. These data together suggest that FMRP regulates the stability of its m6A-marked mRNA targets through YTHDF2, which could potentially contribute to the molecular pathogenesis of FXS. Gene expression analyses were performed on mRNA samples isolated from the cerebral cortex of 6-week-old wild-type and Fmr1 KO male mice. N6-methyladenosine (m6A) epitranscriptome profiling was performed on the same mRNA samples. Six mice were pooled for each biological replicate.N6-甲基腺嘌呤（N6-methyladenosine，m6A）是哺乳动物信使RNA（messenger RNAs，mRNAs）与长链非编码RNA中最为普遍的内部修饰类型。该可逆RNA修饰的生物学功能可通过细胞质与细胞核中的“m6A阅读器（m6A reader）”蛋白进行解读，从而精准调控基因表达过程，例如介导mRNA降解与翻译起始。本研究对成年小鼠大脑皮层的全转录组m6A位点进行了图谱分析，证实m6A是大脑中广泛存在的表观转录组修饰。有趣的是，脆性X智力低下蛋白（fragile X mental retardation protein，FMRP）——一种选择性RNA结合蛋白——的mRNA靶标显著富集m6A修饰位点。功能性FMRP缺失会引发脆性X综合征（Fragile X syndrome，FXS），这是最常见的遗传性智力障碍类型。全转录组基因表达分析显示，在大脑皮层缺失FMRP的情况下，共有2035个基因出现表达差异；且在174个下调的FMRP靶标中，92.5%均带有m6A修饰。生化实验表明，FMRP可结合其mRNA靶标的m6A位点，并以不依赖RNA的方式与m6A阅读器YTHDF2蛋白发生相互作用。FMRP能够维持其mRNA靶标的稳定性，而YTHDF2则会促进这些mRNA的降解。上述数据共同表明，FMRP可通过YTHDF2调控其带有m6A修饰的mRNA靶标的稳定性，这一机制可能参与脆性X综合征的分子病理发生过程。本研究的基因表达分析所用样品，取自6周龄野生型与Fmr1敲除（Fmr1 KO）雄性小鼠的大脑皮层分离得到的mRNA；表观转录组m6A图谱分析则使用了相同的mRNA样品。每个生物学重复均混合6只小鼠的组织样本。