Mechanistic insights into m6A modification of U6 snRNA by human METTL16

Abstract

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.

Figure 1.

The VCR of METTL16 enhances U6 snRNA methylation. (A) Schematic diagrams of human METTL16 and its variants. METTL16_FL: full-length METTL16, MTD: methyltransferase domain, and VCR: vertebrate conserved region. VCR consists of VCR1 (pink) and VCR2 (green) connected by a linker. VCR_ΔL is VCR with the deletion of the linker and was used for crystallization and structural determination. (B) Methylation of U6 snRNA by METTL16_FL and MTD under standard conditions (1 μM of U6 snRNA). (C) Steady-state kinetics of m⁶A modification of U6 snRNA by METTL16_FL and MTD (Supplementary Figure S1A). (D) Gel-shift of U6 snRNA by METTL16_FL (0–0.125 μM) and MTD (0–8 μM) (Supplementary Figure S1B). (E) Methylation of U6 snRNA by METTL16_FL and METTL16_ΔL under standard conditions, as in (B). (F) A stereo view of the structure of VCR_ΔL. Amino acid residues 310–381 and 513–562 are modeled in the structure. VCR1 and VCR2 are colored purple and green, respectively. Bars in the graphs in (B)–(E) are standard deviations (SD) of three independent experiments.

Figure 2.

The VCR is structurally and functionally equivalent to KA1 of TUT1. (A) Sequence alignments of the VCR of human METTL16 and the KA1 domain of human TUT1. The secondary structural elements of the VCR and KA1 domain are indicated above and below the alignments, respectively. (B) A stereo view of the superimposed structures of the VCR of METTL16 (purple) and the KA1 domain of TUT1 (cyan). (C) Topology diagrams of the VCR of METTL16 (left) and the KA1 domain of TUT1 (right). The N- and C-terminal halves are colored pink and green, respectively. (D) Schematic diagrams of METTL16 and its variants used for assays in (E). METTL16_FL: full-length METTL16, MTD: methyltransferase domain, MTD +KA1_wt: a chimeric protein of the N-terminal METTL16 MTD and the C-terminal TUT1 KA1, MTD+KA1_mt: mutant protein of MTD+KA1_wt. The asterisk indicates the R779A/R783A mutation. VCR1 and VCR2 of METTL16 are colored pink and green, respectively. (E) Methylation of U6 snRNA by METTL16_FL and its variants shown in (D), under the conditions in which 50 nM of U6 snRNA was used for the assays. Bars in the graphs are standard deviations (SD) of three independent experiments.

Figure 3.

RNA binding activity of VCR facilitates U6 snRNA methylation. (A) The arginine-rich region (RRR: amino acid residues 382–388) resides between β3 and β4 (left). Conservation analysis of the VCR of METTL16 (right). Conserved and non-conserved residues are colored purple and cyan, respectively (Supplementary Figure S3). (B) Methylation of U6 snRNA by METTL16 variants. Relative activities of METTL16 variants. The activity of METTL_FL under the conditions described in Figure 2E was taken as 1.0. ΔRRR: METTL16 lacking RRR in the VCR. (C) Steady-state kinetics of m⁶A modification of U6 snRNA by METTL16_FL and ΔRRR. (D) Gel shift of U6 snRNA by wild-type VCR (VCR_wt) and its variant lacking RRR, VCR_ΔRRR (0–2.0 μM) (Supplementary Figure S1C). Bars in the graphs in (B)–(D) are standard deviations (SD) of three independent experiments.

Figure 4.

Secondary structure of U6 snRNA for productive catalysis. (A) The secondary structural model of U6 snRNA for methylation by METTL16 (left) is similar to the structure of the MAT2A hairpin (MAT2A_hp1) in the complex with the MTD (right). The MAT2A_hp1 consists of the recognition loop, transition region, and stem required for productive catalysis after complex formation with the MTD. (B) The methylation of U6 snRNA variants with a mutation in the putative transition region, under the standard conditions (1 μM of RNAs). (C) Secondary structures of the U6 snRNA variants used for the assays in (D) and (F). (D) Steady-state kinetics of m⁶A modification of U6 snRNA_Δ5′SL by METTL16_FL and MTD. (E) Steady-state kinetics of m⁶A modification of MAT2A_hp1 by METTL16_FL and MTD (Supplementary Figure S1D). (F) The methylation of U6 snRNA variants by METTL16_FL and MTD under the standard conditions (1 μM of RNAs). Bars in the graphs in (B) and (D)–(F) are standard deviations (SD) of three independent experiments.

Figure 5.

A model of cooperative U6 snRNA methylation by MTD and VCR. (A) Secondary structures of U6 snRNA variants used for assays in (B) and (C). (B) Steady-state kinetics of m⁶A modification of TSmt by METTL16_FL and MTD. (C) Steady-state kinetics of m⁶A modification of ΔTS3 by METTL16_FL and MTD. Bars in the graphs in (B) and (C) are standard deviations (SD) of three independent experiments. (D) A model of the cooperative methylation of U6 snRNA by the MTD and VCR of METTL16. VCR binding to ISL would promote the transition of the structural configurations of telestem-bulge-ISL in U6 snRNA to the bent form and enable the transition structure to be formed in the junction between the telestem and ISL for productive catalysis by the MTD.