Recognition of m6A RNA by the non-canonical reader IMP1

m6A methylation is the prevalent post-transcriptional modification in eukaryotic mRNAs and provides an essential layer of regulation in organismal development and in disease. The information encoded by m6A methylation is integrated into existing RNA regulatory networks by the binding of an expanding list of m6A readers. An important question is how protein readers that do not contain a canonical m6A-specific YTH domain recognize methylated RNA. Here, we show that the non-canonical reader IMP1 directly recognises the m6A group using a dedicated hydrophobic platform in the KH4 domain, creating a stable and high affinity interaction with the methylated RNA targets. Notably, the recognition of the m6A group is independent from the underlying sequence context, but is layered upon IMP1 strong sequence specificity for GGAC RNA. Together, our data indicate that, contrarily to the well-characterised YTH readers, IMP1 recognises and binds both m6A-methylated and non-methylated RNA targets with high affinity. This suggests that m6A methylation does not provide a general layer of control of IMP1 function, but rather plays a directed role in specific regulatory pathways. Overall design: iCLIP in HeLa for IMP1

m6A甲基化（m6A methylation）是真核信使RNA（mRNA）中普遍存在的转录后修饰，在生物体发育及疾病进程中构成关键的调控层面。由m6A甲基化编码的调控信息，可通过持续拓展的m6A结合蛋白（m6A reader）家族的结合作用，整合入现有的RNA调控网络。当前核心科学问题在于：不包含经典m6A特异性YTH结构域（YTH domain）的蛋白结合蛋白如何识别甲基化RNA。本研究证实，非经典结合蛋白IMP1通过其KH4结构域（KH4 domain）中的专属疏水平台直接识别m6A基团，与甲基化RNA靶标形成稳定且高亲和力的相互作用。值得注意的是，IMP1对m6A基团的识别不依赖于其侧翼序列环境，但该识别过程叠加了IMP1对GGAC RNA序列的强特异性结合偏好。综上，我们的研究数据表明，与已被充分表征的YTH家族结合蛋白不同，IMP1能够以高亲和力同时结合m6A甲基化与非甲基化的RNA靶标。这提示m6A甲基化并非对IMP1的功能进行全局性调控，而是在特定调控通路中发挥定向调控作用。
实验整体设计：针对IMP1在HeLa细胞中开展iCLIP实验。