Synaptic plasticity processes, which underlie learning and memory formation, require RNA to be translated local to synapses. The synaptic tagging hypothesis has previously been proposed to explain how mRNAs are available at specific activated synapses. However how RNA is regulated, and which transcripts are silenced or processed as part of the tagging process is still unknown. Modification of RNA by N6-methyladenosine (m⁶A/m) influences the cellular fate of mRNA. Here, by advanced microscopy, we showed that m⁶A demethylation by the eraser protein ALKBH5 occurs at active synaptic ribosomes and at synapses during short term plasticity. We demonstrated that at activated glutamatergic post-synaptic sites, both the YTHDF1 and YTHDF3 reader and the ALKBH5 eraser proteins increase in co-localisation to m⁶A-modified RNAs; but only the readers showed high co-localisation to modified RNAs during late-stage plasticity. The YTHDF1 and YTHFDF3 readers also exhibited differential roles during synaptic maturation suggesting that temporal and subcellular abundance may determine specific function. m⁶A-sequencing of human parahippocampus brain tissue revealed distinct white and grey matter m⁶A methylome profiles indicating that cellular context is a fundamental factor dictating regulated pathways. However, in both neuronal and glial cell-rich tissue, m⁶A effector proteins are themselves modified and m⁶A epitranscriptional and posttranslational modification processes coregulate protein cascades. We hypothesise that the availability m⁶A effector protein machinery in conjunction with RNA modification, may be important in the formation of condensed synaptic nanodomain assemblies through liquid-liquid phase separation. Our findings support that m⁶A demethylation by ALKBH5 is an intrinsic component of the synaptic tagging hypothesis and a molecular switch which leads to alterations in the RNA methylome, synaptic dysfunction and potentially reversible disease states.

突触可塑性过程是学习和记忆形成的基础，需要将 RNA 在局部翻译成突触。先前已提出突触标记假说来解释 mRNA 如何在特定激活的突触处可用。然而，RNA 是如何被调节的，哪些转录本被沉默或作为标记过程的一部分被处理仍然未知。N6-甲基腺苷 (m ⁶ A/m) 对 RNA 的修饰影响 mRNA 的细胞命运。在这里，通过高级显微镜，我们发现 m ⁶橡皮擦蛋白 ALKBH5 的去甲基化发生在活跃的突触核糖体和短期可塑性期间的突触处。我们证明，在激活的谷氨酸能突触后位点，YTHDF1 和 YTHDF3 读取器以及 ALKBH5 擦除蛋白与 m ⁶ A 修饰的 RNA 的共定位增加；但只有读者在后期可塑性期间表现出与修饰 RNA 的高度共定位。YTHDF1 和 YTHFDF3 阅读器在突触成熟过程中也表现出不同的作用，这表明时间和亚细胞丰度可能决定特定功能。m ⁶人海马旁回脑组织的 A 序列显示明显的白质和灰质 m ⁶甲基化组概况表明细胞环境是决定调节途径的基本因素。然而，在富含神经元和神经胶质细胞的组织中，m ⁶ A 效应蛋白本身被修饰，并且 m ⁶ A 外转录和翻译后修饰过程共同调节蛋白质级联。我们假设可用性 m ⁶ A 效应蛋白机制与 RNA 修饰相结合，可能对通过液-液相分离形成浓缩突触纳米域组件很重要。我们的研究结果支持 m ⁶ALKBH5 的去甲基化是突触标记假说的内在组成部分，也是导致 RNA 甲基化组改变、突触功能障碍和潜在可逆疾病状态的分子开关。