Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN, 46556, USA.
Synchrotron Radiation Research Section of MCL, National Cancer Institute, Argonne, IL, 60439, USA.
Sci Rep. 2018 Mar 28;8(1):5311. doi: 10.1038/s41598-018-23608-8.
N-methyladenosine (mA) is an abundant modification in messenger RNA and noncoding RNAs that affects RNA metabolism. Methyltransferase-like protein 16 (METTL16) is a recently confirmed mA RNA methyltransferase that methylates U6 spliceosomal RNA and interacts with the 3'-terminal RNA triple helix of MALAT1 (metastasis-associated lung adenocarcinoma transcript 1). Here, we present two X-ray crystal structures of the N-terminal methyltransferase domain (residues 1-291) of human METTL16 (METTL16_291): an apo structure at 1.9 Å resolution and a post-catalytic S-adenosylhomocysteine-bound complex at 2.1 Å resolution. The structures revealed a highly conserved Rossmann fold that is characteristic of Class I S-adenosylmethionine-dependent methyltransferases and a large, positively charged groove. This groove likely represents the RNA-binding site and it includes structural elements unique to METTL16. In-depth analysis of the active site led to a model of the methyl transfer reaction catalyzed by METTL16. In contrast to the major mA methyltransferase heterodimer METTL3/METTL14, full-length METTL16 forms a homodimer and METTL16_291 exists as a monomer based on size-exclusion chromatography. A native gel-shift assay shows that METTL16 binds to the MALAT1 RNA triple helix, but monomeric METTL16_291 does not. Our results provide insights into the molecular structure of METTL16, which is distinct from METTL3/METTL14.
N6-甲基腺苷（m6A）是信使 RNA 和非编码 RNA 中丰富的修饰物，影响 RNA 代谢。甲基转移酶样蛋白 16（METTL16）是一种新确认的 m6A RNA 甲基转移酶，它甲基化 U6 剪接体 RNA 并与 MALAT1（转移相关的肺腺癌转录本 1）的 3'-末端 RNA 三螺旋相互作用。在这里，我们呈现了人 METTL16（METTL16_291）的 N 端甲基转移酶结构域（残基 1-291）的两个 X 射线晶体结构：一个无配体结构分辨率为 1.9 Å，一个后催化 S-腺苷同型半胱氨酸结合复合物分辨率为 2.1 Å。这些结构揭示了一个高度保守的 Rossmann 折叠，这是 I 类 S-腺苷甲硫氨酸依赖性甲基转移酶的特征，以及一个大的、带正电荷的凹槽。这个凹槽可能代表 RNA 结合位点，它包括 METTL16 特有的结构元素。对活性位点的深入分析导致了由 METTL16 催化的甲基转移反应的模型。与主要的 m6A 甲基转移酶异二聚体 METTL3/METTL14 不同，全长 METTL16 形成同源二聚体，而根据大小排阻层析，METTL16_291 作为单体存在。天然凝胶迁移实验表明 METTL16 结合 MALAT1 RNA 三螺旋，但单体 METTL16_291 不结合。我们的结果提供了 METTL16 分子结构的见解，它与 METTL3/METTL14 不同。
