Effect of METTL1 KO on the binding ability of QKI7

N7-methylguanosine (m7G) modification, routinely occurring at the 5' cap of mRNA or within tRNA and rRNA, also exists internally in mRNA. Although essential for mRNA translation as well as stress response, the “reader” protein for mRNA internal m7G modification is still unrevealed. Here, we reported that Quaking protein (QKI), especially QKI7, can selectively recognize the internal mRNA m7G decoration in the cytosol of various cell types. We identified over 1000 confident m7G-modified and QKI binding RNA targets with a conserved motif, “GANGAN (N=A/U/G)”. More strikingly, internal m7G reader QKI7 directly interacts with the SG core protein G3BP1 and can shuttle a subset of m7G-modified transcripts into SG mRNA pool under oxidative stress condition. Additionally, by sequestering mRNA within SGs, QKI7 modulates the translation efficiency of selected transcripts. Moreover, in line with the observation that doxorubicin triggers the assembly of SGs, QKI7 mediates the sensitivity of cancer cells to chemotherapy drug treatment. Overall design: To study whether METTL1 mediates the binding ability between QKI7 and its target transcripts, RNA immunoprecipitation sequencing (RIP-seq) was conducted in HepG2-cas9 cells stably expressing QKI7 protein with or without METTL1 KO. Briefly, HepG2-cas9 cells were infected with lentivirus, pmiRNA1-3 x Flag-QKI7. After that, METTL1 was depleted using sgRNA. Only the GFP-positive cells were used for study and expanded in DMEM medium. RIP assay was performed with Flag antibody (F3165, Sigma).

N7-甲基鸟苷（N7-methylguanosine, m7G）修饰常规存在于信使RNA（mRNA）的5'帽结构、转运RNA（tRNA）与核糖体RNA（rRNA）中，同时也可分布于mRNA内部。尽管该修饰对mRNA翻译及应激反应均至关重要，但目前尚未发现介导mRNA内部m7G修饰的读取器蛋白。
本研究证实，Quaking蛋白（Quaking protein, QKI），尤其是QKI7亚型，可在多种细胞类型的胞质中选择性识别mRNA内部的m7G修饰。我们通过保守基序"GANGAN (N=A/U/G)"，筛选得到了超过1000个兼具m7G修饰与QKI结合特性的可信RNA靶标。更引人注目的是，作为内部m7G读取器的QKI7可与应激颗粒（stress granule, SG）核心蛋白G3BP1直接相互作用，并能在氧化应激条件下，将部分携带m7G修饰的转录本转运至应激颗粒内的mRNA库中。此外，QKI7通过将mRNA捕获至应激颗粒中，调控特定转录本的翻译效率。进一步结合多柔比星（doxorubicin）可诱导应激颗粒组装的研究结论，我们发现QKI7可调控癌细胞对化疗药物的敏感性。
整体实验设计：为探究METTL1是否介导QKI7与其靶标转录本的结合能力，我们在稳定表达QKI7蛋白的HepG2-cas9细胞中设置METTL1敲除（knockout, KO）与未敲除两组，开展RNA免疫共沉淀测序（RNA immunoprecipitation sequencing, RIP-seq）。具体步骤如下：先将慢病毒pmiRNA1-3×Flag-QKI7感染HepG2-cas9细胞，随后通过单导RNA（single guide RNA, sgRNA）敲除METTL1基因；仅选取GFP阳性细胞进行后续研究，并在达尔伯克改良伊格尔培养基（Dulbecco's Modified Eagle Medium, DMEM）中扩增培养。实验采用Flag抗体（F3165，Sigma）完成RNA免疫共沉淀操作。