mRNA circularization by METTL3-eIF3h enhances translation

N6-methyladenosine (m6A) modification of mRNA catalyzed by METTL3 is enriched at a subset of stop codons. METTL3 can promote translation but the mechanism and widespread relevance remain unknown. Here we show that METTL3 enhances translation only when tethered to reporter mRNA at sites close to the stop codon supporting a mRNA looping mechanism for ribosome recycling and translational control. Electron microscopy revealed the topology of individual polyribosomes with single METTL3 foci found in close proximity to 5' cap-binding proteins. We identify a direct physical and functional interaction between METTL3 and the eukaryotic translation initiation factor 3 subunit h (eIF3h). METTL3 promotes translation of a large subset of oncogenic mRNAs, including BRD4 that are also m6A-modified in human primary lung tumors. The METTL3-eIF3h interaction is required for enhanced translation, formation of densely packed polyribosomes, and oncogenic transformation. METTL3 depletion inhibits tumorigenicity and sensitizes lung cancer cells to BRD4 inhibition. These findings uncover a mRNA looping mechanism of translation control and identify METTL3-eIF3h as a potential cancer therapeutic target. Overall design: METTL3 expression was depleted in HeLa cells using lentivirus mediated shRNA knockdown. The METTL3 knockdown cells and the control cells were subjected to sucrose gradient ultracentifuge for the polysome profiling. The RNA samples isolated from the polysome fractions and subpolysome fractions were used for high throughput sequencing for the analysis of translation efficiency. For the analysis of mRNA stability, METTL3 knockdown cells and the control cells were treated with ActD for different time points then the RNA samples were isolated for high throughput sequencing and the subsequent calculation of mRNA halflife.N6-甲基腺嘌呤（N6-methyladenosine, m6A）修饰是由METTL3催化的mRNA修饰，该修饰富集于部分终止密码子区域。METTL3可促进翻译过程，但其具体调控机制与广泛生物学相关性仍未明确。本研究证实，仅当METTL3被锚定至靠近终止密码子的报告mRNA位点时，方能增强翻译效率，这一结果支持了核糖体循环与翻译调控的mRNA环化机制。电子显微镜观测揭示了单个多聚核糖体的拓扑结构，且发现单个METTL3焦点与5'帽结合蛋白的空间位置极为接近。本研究鉴定出METTL3与真核翻译起始因子3亚基h（eIF3h）之间存在直接的物理与功能相互作用。METTL3可促进大量致癌mRNA的翻译，其中包括在人类原发性肺肿瘤中存在m6A修饰的BRD4。METTL3与eIF3h的相互作用是增强翻译效率、形成致密堆积多聚核糖体以及介导致癌转化所必需的。敲低METTL3可抑制肿瘤发生能力，并使肺癌细胞对BRD4抑制剂更为敏感。本研究揭示了一种基于mRNA环化的翻译调控机制，并将METTL3-eIF3h鉴定为潜在的癌症治疗靶点。实验整体设计：采用慢病毒介导的短发夹RNA（shRNA）敲低HeLa细胞中的METTL3表达。将METTL3敲低细胞与对照细胞进行蔗糖梯度超速离心，以开展多聚核糖体谱分析。从多聚核糖体组分及亚多聚核糖体组分中分离得到的RNA样本，将用于高通量测序以分析翻译效率。在mRNA稳定性分析中，用放线菌素D（ActD）分别处理METTL3敲低细胞与对照细胞不同时长，随后分离RNA样本进行高通量测序，并据此计算mRNA的半衰期。