Catalytic activity of nuclear METTL3 promotes functional m6A modifications that drive retinal development [Nanopore-seq]. Catalytic activity of nuclear METTL3 promotes functional m6A modifications that drive retinal development [Nanopore-seq]

METTL3 catalyzes N6-methyladenosine (m6A) on mRNA, regulating RNA metabolism, but its role in early retinal development remains virtually unstudied. Using stem cell-derived 3D retinal organoids to model retinal progenitor cell (RPC) differentiation, we found that loss of METTL3 nuclear m6A enzymatic activity impairs formation of the Rx+ retinal anlage in vitro. Through dCas13b-FTO m6A engineering, we demonstrate that m6A modifications at the Six3 3'UTR control its stability. While Mettl3 loss altered histone modifications, direct METTL3 chromatin targets were not transcriptionally functional. Integration of transcriptome-wide m6A and protein-RNA mapping with a degron-based strategy revealed immediate effects of METTL3 degradation in RPCs, uncovering a regulatory interaction between METTL3 and its RNA target Ythdf1. We demonstrate uncoupling of chromatin accessibility from changes in retinal transcription and m6A modifications. In vivo studies of Mettl3-deficient RPCs showed altered cell cycle and impaired retinal ganglion cell generation. Our findings establish METTL3-dependent m6A modifications as an essential post-transcriptional layer that drives retinal development. Overall design: Nanopore-seq profiling of day 6 retinal organoids of WT Rx:GFP. Direct RNA libraries were made by RNA002 kit.

METTL3可催化mRNA上的N6-甲基腺苷（N6-methyladenosine，m6A）修饰，调控RNA代谢，但其在视网膜早期发育中的作用几乎尚未得到研究。本研究借助干细胞来源的三维视网膜类器官模拟视网膜祖细胞（retinal progenitor cell，RPC）的分化过程，发现METTL3细胞核m6A酶活性缺失会在体外损害Rx阳性视网膜原基的形成。通过dCas13b-FTO介导的m6A编辑技术，本研究证实Six3基因3'非翻译区（3'UTR）的m6A修饰可调控其mRNA稳定性。尽管Mettl3基因缺失会改变组蛋白修饰，但METTL3的直接染色质靶标并未表现出转录活性。将全转录组m6A分析、蛋白质-RNA结合图谱与基于降解标签（degron）的策略相结合，本研究揭示了METTL3降解在RPCs中的即时效应，并发现了METTL3与其RNA靶标Ythdf1之间的调控互作关系。本研究证实染色质开放性与视网膜转录及m6A修饰的变化之间存在解偶联现象。对Mettl3基因缺失的RPCs开展体内研究发现，其细胞周期发生改变，视网膜神经节细胞的生成受到损害。本研究结果证实，依赖于METTL3的m6A修饰是驱动视网膜发育的关键转录后调控层级。整体实验设计：对野生型Rx:GFP的第6天视网膜类器官开展纳米孔测序（Nanopore-seq）分析，采用RNA002试剂盒构建直接RNA文库。