Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba 277-8562, Japan.
Nucleic Acids Res. 2020 May 21;48(9):5157-5168. doi: 10.1093/nar/gkaa227.
The N6-methyladenosine modification at position 43 (m6A43) of U6 snRNA is catalyzed by METTL16, and is important for the 5'-splice site recognition by U6 snRNA during pre-mRNA splicing. Human METTL16 consists of the N-terminal methyltransferase domain (MTD) and the C-terminal vertebrate conserved region (VCR). While the MTD has an intrinsic property to recognize a specific sequence in the distinct structural context of RNA, the VCR functions have remained uncharacterized. Here, we present structural and functional analyses of the human METTL16 VCR. The VCR increases the affinity of METTL16 toward U6 snRNA, and the conserved basic region in VCR is important for the METTL16-U6 snRNA interaction. The VCR structure is topologically homologous to the C-terminal RNA binding domain, KA1, in U6 snRNA-specific terminal uridylyl transferase 1 (TUT1). A chimera of the N-terminal MTD of METTL16 and the C-terminal KA1 of TUT1 methylated U6 snRNA more efficiently than the MTD, indicating the functional conservation of the VCR and KA1 for U6 snRNA biogenesis. The VCR interacts with the internal stem-loop (ISL) within U6 snRNA, and this interaction would induce the conformational rearrangement of the A43-containing region of U6 snRNA, thereby modifying the RNA structure to become suitable for productive catalysis by the MTD. Therefore, the MTD and VCR in METTL16 cooperatively facilitate the m6A43 U6 snRNA modification.
U6 snRNA 第 43 位的 N6-甲基腺苷修饰（m6A43）由 METTL16 催化，对于 U6 snRNA 在 pre-mRNA 剪接过程中识别 5'-剪接位点非常重要。人 METTL16 由 N 端甲基转移酶结构域（MTD）和 C 端脊椎动物保守区（VCR）组成。虽然 MTD 具有在 RNA 独特结构环境中识别特定序列的内在特性，但 VCR 的功能仍未被阐明。在这里，我们对人 METTL16 VCR 的结构和功能进行了分析。VCR 增加了 METTL16 对 U6 snRNA 的亲和力，VCR 中的保守碱性区域对 METTL16-U6 snRNA 相互作用很重要。VCR 的结构在拓扑上与 U6 snRNA 特异性末端尿苷酰转移酶 1（TUT1）的 C 端 RNA 结合结构域 KA1 同源。METTL16 的 N 端 MTD 和 TUT1 的 C 端 KA1 的嵌合体比 MTD 更有效地甲基化 U6 snRNA，表明 VCR 和 KA1 在 U6 snRNA 生物发生中的功能保守性。VCR 与 U6 snRNA 内的茎环（ISL）相互作用，这种相互作用会诱导 U6 snRNA 中含 A43 区域的构象重排，从而改变 RNA 结构，使其适合 MTD 的有效催化。因此，METTL16 中的 MTD 和 VCR 协同促进 m6A43 U6 snRNA 修饰。
