Identification and Characterization of the Mammalian Nuclear RNA N6-Adenosine Methyltransferase Core Complex

N6-methyladenosine (m6A) is the most prevalent internal modification found in mammalian messenger and non-coding RNAs. The discoveries of functionally significant demethylases that reverse this methylation as well as the recently revealed m6A distributions in mammalian transcriptomes strongly indicate regulatory functions of this modification. Here we report the identification and characterization of the mammalian nuclear RNA N6-adenosine methyltransferase core (RNMTC) complex. Besides METTL3, a methyltransferase which was the only known component of RNMTC in the past, we discovered that a previously uncharacterized methyltransferase, METTL14, exhibits a N6-adenosine methyltransferase activity higher than METTL3. Together with WTAP, the third component that dramatically affects the cellular m6A level, these three proteins form the core complex that orchestrates m6A deposition on mammalian nuclear RNA. Biochemistry assays, imaging experiments, as well as transcriptome-wide analyses of the binding sites and their effects on m6A methylation support methylation function and reveal new insights of RNMTC. Overall design: PAR-CLIP and m6A-seq in HeLa cells

N6-甲基腺嘌呤（N6-methyladenosine, m6A）是哺乳动物信使RNA与非编码RNA中最为普遍的内部修饰类型。可逆转该甲基化修饰的功能性去甲基化酶的发现，以及近期揭示的哺乳动物转录组内m6A的分布特征，均强烈提示该修饰具备调控功能。本研究完成了哺乳动物细胞核RNA N6-腺嘌呤甲基转移酶核心（RNMTC）复合物的鉴定与表征：除甲基转移酶样3（METTL3）这一既往唯一已知的RNMTC组分外，我们还发现此前未被表征的甲基转移酶METTL14的N6-腺嘌呤甲基转移酶活性高于METTL3。结合可显著影响细胞m6A水平的第三组分WTAP，这三种蛋白质共同构成了调控哺乳动物细胞核RNA上m6A修饰沉积的核心复合物。生化检测实验、成像实验以及全转录组水平的结合位点分析与m6A甲基化影响分析，验证了该复合物的甲基化功能，并为RNMTC的作用机制提供了全新见解。实验设计：在HeLa细胞中开展PAR-CLIP与m6A测序实验。