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

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: GLORI m6A-seq profiling of dTAG13 induced acute degradation of METTL3 in day 6 retinal organoids of Rx:GFP harboring FKBP12F36V-HA knocking into c-termincal of endogenous Mettl3.

METTL3可催化信使RNA（messenger RNA，mRNA）上的N6-甲基腺嘌呤（N6-methyladenosine，m6A）修饰，调控RNA代谢，但目前学界对其在视网膜早期发育中的作用几乎尚无研究。我们利用干细胞来源的3D视网膜类器官模拟视网膜祖细胞（retinal progenitor cell，RPC）分化过程，发现METTL3的细胞核m6A酶活性缺失会在体外抑制Rx阳性视网膜原基的形成。通过dCas13b-FTO介导的m6A编辑系统，我们证实Six3基因3'非翻译区（3' untranslated region，3'UTR）的m6A修饰可调控其RNA稳定性。尽管Mettl3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。我们将全转录组m6A测序、蛋白-RNA互作图谱与基于降解标签（degron）的实验策略相结合，揭示了METTL3降解在视网膜祖细胞中的即时效应，并发现了METTL3与其RNA靶标Ythdf1之间的调控互作关系。我们证实染色质可及性与视网膜转录及m6A修饰变化之间存在解偶联现象。对Mettl3缺陷型视网膜祖细胞的体内研究显示，其细胞周期发生改变，且视网膜神经节细胞生成受损。本研究证实，依赖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：对在内源Mettl3基因C端敲入FKBP12F36V-HA标签的Rx:GFP转基因小鼠第6天视网膜类器官，经dTAG13诱导METTL3发生急性降解后，开展GLORI m6A测序分析。