Abstract N 6-methyladenosine (m6A) is the most abundant modification in mRNA. The core of the human N 6-methyltransferase complex (MTC) is formed by a heterodimer consisting of METTL3 and METTL14, which specifically catalyzes m6A formation within an RRACH sequence context. Using recombinant proteins in a site-specific methylation assay that allows determination of quantitative methylation yields, our results show that this complex methylates its target RNAs not only sequence but also secondary structure dependent. Furthermore, we demonstrate the role of specific protein domains on both RNA binding and substrate turnover, focusing on postulated RNA binding elements. Our results show that one zinc finger motif within the complex is sufficient to bind RNA, however, both zinc fingers are required for methylation activity. We show that the N-terminal domain of METTL3 alters the secondary structure dependence of methylation yields. Our results demonstrate that a cooperative effect of all RNA-binding elements in the METTL3–METTL14 complex is required for efficient catalysis, and that binding of further proteins affecting the NTD of METTL3 may regulate substrate specificity.

中文翻译：

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METTL3-METTL14 mRNA 甲基化中的 RNA 二级结构依赖性受 METTL3 的 N 端结构域调节

摘要 N 6-甲基腺苷 (m6A) 是 mRNA 中最丰富的修饰。人类 N 6-甲基转移酶复合物 (MTC) 的核心由由 METTL3 和 METTL14 组成的异二聚体形成，其在 RRACH 序列环境中特异性催化 m6A 形成。在位点特异性甲基化分析中使用重组蛋白，可以确定定量甲基化产量，我们的结果表明，这种复合物不仅序列而且二级结构依赖于其目标 RNA 的甲基化。此外，我们证明了特定蛋白质域对 RNA 结合和底物周转的作用，重点是假定的 RNA 结合元件。我们的结果表明复合物中的一个锌指基序足以结合 RNA，然而，甲基化活性需要两个锌指。我们表明 METTL3 的 N 端结构域改变了甲基化产量的二级结构依赖性。我们的结果表明，METTL3-METTL14 复合物中所有 RNA 结合元件的协同作用是有效催化所必需的，并且影响 METTL3 的 NTD 的其他蛋白质的结合可能会调节底物特异性。