Genome Institute of Singapore, 60 Biopolis Street, Singapore 138672, Singapore.
School of Biological Sciences, Nanyang Technological University, Singapore 637551, Singapore.
Nucleic Acids Res. 2020 Sep 18;48(16):9250-9261. doi: 10.1093/nar/gkaa684.
N 6-methylation of 2'-O-methyladenosine (Am) in RNA occurs in eukaryotic cells to generate N6,2'-O-dimethyladenosine (m6Am). Identification of the methyltransferase responsible for m6Am catalysis has accelerated studies on the function of m6Am in RNA processing. While m6Am is generally found in the first transcribed nucleotide of mRNAs, the modification is also found internally within U2 snRNA. However, the writer required for catalyzing internal m6Am formation had remained elusive. By sequencing transcriptome-wide RNA methylation at single-base-resolution, we identified human METTL4 as the writer that directly methylates Am at U2 snRNA position 30 into m6Am. We found that METTL4 localizes to the nucleus and its conserved methyltransferase catalytic site is required for U2 snRNA methylation. By sequencing human cells with overexpressed Mettl4, we determined METTL4's in vivo target RNA motif specificity. In the absence of Mettl4 in human cells, U2 snRNA lacks m6Am thereby affecting a subset of splicing events that exhibit specific features such as 3' splice-site weakness and an increase in exon inclusion. These findings suggest that METTL4 methylation of U2 snRNA regulates splicing of specific pre-mRNA transcripts.
RNA 中 2'-O-甲基腺苷（Am）的 N6-甲基化发生在真核细胞中，以生成 N6,2'-O-二甲基腺苷（m6Am）。鉴定负责 m6Am 催化的甲基转移酶加速了对 m6Am 在 RNA 加工中的功能的研究。虽然 m6Am 通常在 mRNA 的第一个转录核苷酸中发现，但该修饰也存在于 U2 snRNA 内部。然而，催化内部 m6Am 形成的书写器仍然难以捉摸。通过在单碱基分辨率上对转录组范围的 RNA 甲基化进行测序，我们确定人类 METTL4 是直接将 U2 snRNA 位置 30 处的 Am 甲基化为 m6Am 的书写器。我们发现 METTL4 定位于细胞核中，其保守的甲基转移酶催化位点是 U2 snRNA 甲基化所必需的。通过对过表达 Mettl4 的人类细胞进行测序，我们确定了 METTL4 在体内靶 RNA 基序特异性。在人类细胞中缺乏 Mettl4 的情况下，U2 snRNA 缺乏 m6Am，从而影响了一组具有特定特征的剪接事件，例如 3'剪接位点减弱和外显子包含增加。这些发现表明，U2 snRNA 的 METTL4 甲基化调节特定前体 mRNA 转录本的剪接。
